3D (three-dimensional) transplantation material system with nerve protection function and application thereof
阅读说明：本技术 一种具有神经保护功能的3d立体移植材料体系及其应用 (3D (three-dimensional) transplantation material system with nerve protection function and application thereof ) 是由 胡荣 方煊宇 张超 张旭阳 周腾渊 张水仙 刘丹 葛红飞 于 2020-12-08 设计创作，主要内容包括：本发明提供了一种具有神经保护功能的3D立体移植材料体系及其应用,属于脑损伤药物技术领域。褪黑素在制备针对在缺血缺氧条件下的神经干细胞的神经保护的药物中的应用。本发明还提供了一种具有神经保护功能的3D立体移植体系包括褪黑素、神经干细胞和基质胶。本发明通过3D移植策略能明显促进损伤模型大鼠的神经功能恢复,其中褪黑素为神经干细胞适应损伤灶不利微环境提供了保护作用,基质胶的加入可以从立体层面给予模型损伤后形成的脑组织空腔提供结构支持,提供细胞生长的立体环境,加强细胞修复效果,从基础研究层面为以后颅脑损伤的临床治疗策略的发展提供了一个方向。(The invention provides a 3D (three-dimensional) transplantation material system with a nerve protection function and application thereof, belonging to the technical field of brain injury medicines. Use of melatonin for the preparation of a medicament for neuroprotection against neural stem cells under conditions of ischemia and hypoxia. The invention also provides a 3D three-dimensional transplantation system with a nerve protection function, which comprises melatonin, neural stem cells and matrigel. The 3D transplantation strategy can obviously promote the nerve function recovery of the injured model rat, wherein the melatonin provides a protective effect for the adaptation of the nerve stem cells to the adverse microenvironment of the injured focus, and the addition of the matrigel can provide structural support for the brain tissue cavity formed after the injury of the model from the three-dimensional level, provide the three-dimensional environment for cell growth, enhance the cell repair effect, and provide a direction for the development of the clinical treatment strategy of the craniocerebral injury from the basic research level.)
1. Use of melatonin for the preparation of a medicament for neuroprotection against neural stem cells under conditions of ischemia and hypoxia.
2. The use of claim 1, wherein the neuroprotection is manifested by melatonin's ability to render neural stem cells viable under hypoxic and ischemic conditions.
3. A graft material having a neuroprotective function, comprising melatonin and neural stem cells.
4. The graft material with neuroprotective effect according to claim 3, wherein the final concentration of melatonin solution in the graft material is not less than 25 μmol/L, and the concentration of said neural stem cells is 105～107/ml。
5. Use of a graft material according to claim 3 or 4 in the manufacture of a medicament for the recovery of neurological function.
6. Use of the graft material of claim 3 or 4 in the manufacture of a medicament for the treatment of craniocerebral injury.
7. A3D stereotactic implant system having craniocerebral injury repair function, comprising the implant material of claim 3 or 4 and a matrigel.
8. Use of the 3D stereotactic implant system of claim 7 in the manufacture of a medicament for treating craniocerebral injury.
9. Use of a 3D stereotactic system according to claim 7 for the preparation of a medicament for the recovery of neurological function.
Technical Field
The invention belongs to the technical field of brain injury medicines, and particularly relates to a 3D (three-dimensional) transplantation material system with a nerve protection function and application thereof.
Background
Craniocerebral injury is the most common injury form in modern war injuries, the incidence rate is up to more than 20%, the death rate is the first of various injuries, and timely and effective treatment measures are still limited up to now. Although the survival rate of patients is obviously improved by the widely applied craniocerebral injury acute-stage operation treatment scheme at present, the remaining nerve dysfunction such as paraplegia, cognition and the like still has the core problem of influencing the life quality of survived patients. Therefore, it is currently the leading line of research in this field to find strategies and methods to promote repair of craniocerebral injuries.
Neural stem cells are a type of pluripotent stem cells that have a self-renewal ability and can differentiate into neurons, glial cells, oligodendrocytes, and the like, found in brain tissues of various animals. Under normal conditions, neural stem cells are in low stock and resting state in adult animals. Modern research shows that under the condition of lesions, neural stem cells can be activated and participate in nerve repair of focal areas, so that the possibility of using the neural stem cells as a repair treatment means in craniocerebral lesions is valued by people.
Neural stem cell therapy has been a focus of attention. However, the application of the endogenous neural stem cells is limited due to the small number of the endogenous neural stem cells, and the transplantation of the exogenous neural stem cells can be a method for breaking through the problem. Supplementation with exogenous cells has been essentially a routine step in medical research. Cell transplantation has greater compatibility and greater choice of sources than tissue transplantation. And the applicability of the stem cells is greatly improved due to the proliferation and differentiation capacity of the stem cells. Numerous studies have shown that neural stem cells have achieved considerable success in repair and therapy. Baker successfully carries out neural stem cell transplantation treatment in a pig brain stroke model in 2017, and research and detection show that after transplantation, the tissue function is obviously recovered, the number of microglia in a focus area is reduced, migration of neural stem cells is increased, and the neural stem cells are differentiated into neurons and oligodendrocytes to integrate a neural network. In vitro culture, the neurons with directionally differentiated dopamine phenotype are also applied to a Parkinson disease model, good recovery effect is achieved aiming at complex craniocerebral injury conditions, and although a stem cell transplantation treatment scheme is already in consensus, a plurality of different constant schemes still exist, such as bone marrow injection, ventricle transplantation, hippocampal transplantation, intravenous injection, cerebral pseudo-surgery and other different schemes. Different transplantation treatment schemes can finally verify the promotion effect of the neural stem cell transplantation on the repair of the nerve function damage. In 2018, AliJahanbaziJahan-Abad transplanted human neural stem/progenitor cells (hNS/PCs) isolated from the brain of an epileptic during surgery into a rat model of craniocerebral injury. The experiment can effectively reduce the size of a lesion area, inhibit the nerve inflammation after injury, improve the nerve function recovery of a rat and reduce the gliosis of the injury part.
Melatonin is a hormone secreted by the pineal body at night under the influence of normal circadian rhythm control, and melatonin or its metabolites can be detected in plasma, saliva, urine, and the like. Melatonin can influence molecular signal pathways (such as Notch, MAPK and the like) by regulating ion channels (such as calcium ion channels) in cells, and accurately regulate physiological activities and life cycle of cells by regulating mitochondrial respiration and other ways. A small amount of literature indicates that melatonin can promote proliferation of neural stem cells, but the influence on the biological behavior of the neural stem cells and the damage of the nervous system caused by combined transplantation of the melatonin and the neural stem cells is less researched at present.
Disclosure of Invention
In view of the above, the present invention aims to provide a new use of melatonin, namely, an application of melatonin in preparing a medicament for neuroprotection of neural stem cells under ischemia and hypoxia conditions.
The invention also aims to provide a transplantation material with a nerve protection function and application thereof, and the transplantation material has nerve function recovery when being transplanted into a craniocerebral injury model.
The invention also aims to provide a 3D stereo-transplantation system with the function of repairing craniocerebral injury and application thereof, wherein the 3D stereo-transplantation system obviously promotes the recovery of the nerve function of an injured model rat.
The invention provides an application of melatonin in preparing a medicament for protecting nerve stem cells under an ischemic and anoxic condition.
Preferably, the neuroprotection is manifested in that melatonin enables survival of neural stem cells under ischemic and hypoxic conditions.
The invention provides a transplantation material with a nerve protection function, which comprises melatonin and neural stem cells.
Preferably, in the graft material, the final concentration of the melatonin solution is not less than 25. mu. mol/L, and the concentration of the neural stem cells is 105～107/ml。
The invention provides application of the transplantation material in preparing a medicine for recovering the nerve function.
The invention provides application of the transplantation material in preparing a medicine for treating craniocerebral injury.
The invention provides a 3D (three-dimensional) transplantation system with a craniocerebral injury repair function, which comprises a transplantation material and matrigel.
The invention provides an application of the 3D stereo transplantation system in preparing a medicine for treating craniocerebral injury.
The invention provides an application of the 3D stereo-transplantation system in preparing a medicine for recovering the nerve function
Adding melatonin with different gradient concentrations into the culture solution, observing the morphological change of the neural stem cells, and determining the cell proliferation capacity and the immunofluorescence staining to identify the differentiation capacity by adopting a CCK8 method. The results show that the immunofluorescence identifies the neural stem cells (antibodies), and the potential of the melatonin to differentiate the neural stem cells into neural sub-cells such as astrocytes (GFAP), neurons (Nestin), oligodendrocytes (Olig2) and the like is determined.
The invention provides an application of melatonin in preparing a medicament for protecting nerve stem cells under an ischemic and anoxic condition. The invention is prepared by the method of containing cobalt chloride (CoCl)2) The EBSS sugar-free culture medium simulates the anoxic and sugar-deficient condition of a lesion area after injury, and researches the influence of melatonin on the viability of the neural stem cells, and the results show that the viability of the neural stem cells of an untreated group is greatly reduced, and the survival quantity of the neural stem cells is obviously increased compared with that of the untreated group after the melatonin is treated by 25 mu mol/L, so that the viability of the neural stem cells is improved. This indicates that melatonin has a better nerve protection effect on neural stem cells under the conditions of ischemia and hypoxia.
The transplantation material with the nerve protection function comprises melatonin and neural stem cells. In view of the potential that the neural stem cells can activate the neural restoration function of a focal region and differentiate towards neural sub-class cells, the experiment of the invention proves that melatonin is beneficial to improving the survival capability of the neural stem cells under the condition of blood deficiency and oxygen deficiency, so that the neural stem cells can realize the differentiation towards functional neurons and other types of neural cells under the action of the melatonin by transplanting the material to the focus part of the brain, thereby achieving the purpose of neural restoration in the brain injury.
The 3D three-dimensional transplantation system with the craniocerebral injury repair function provided by the invention comprises the transplantation material and the matrigel. The 3D three-dimensional transplantation system containing melatonin, neural stem cells and matrigel can obviously promote the recovery of the nerve function of a damaged model rat through a 3D transplantation strategy, wherein the melatonin provides a protection effect for the neural stem cells to adapt to the adverse microenvironment of a damaged focus, and the matrigel can provide structural support for a brain tissue cavity formed after the model is damaged from a three-dimensional layer, provide a three-dimensional environment for cell growth and enhance the cell repair effect. The invention provides a direction for the development of clinical treatment strategies for the later craniocerebral injuries from the basic research level.
Drawings
FIG. 1 is an observation diagram of a neural stem cell culture state under observation of an inverted phase contrast microscope, and FIG. 1A is a diagram of a neural stem cell morphology isolated and extracted from a frontal cortex; fig. 1B is a picture of proliferation and aggregation of neural stem cells into spheres after three days of suspension culture, with a scale: 50 μm;
FIG. 2 shows the identification result of immunofluorescence staining of the specific molecular markers Nestin and SOX2 of neural stem cells, and the scale: 50 μm;
FIG. 3 is a graph showing the result of the determination of the multipotentiality of neural stem cells, wherein FIG. 3A is a graph showing the DCX-labeling of neural stem cells to differentiate into neuroblasts (neuralbolasts); FIG. 3B shows the identification of neural stem cell differentiation into oligodendrocytes by Oligo2 marker; FIG. 3C shows the GFAP marker identifying differentiation of neural stem cells into astrocytes and DAPI counterstain nuclei, all at 50 μm;
FIG. 4 shows the use of a catalyst containing CoCl2The OGD model was established in EBSS medium of (1), in which fig. 4A: normal neural stem cell culture controls; FIG. 4B: processing an OGD model for 2 h; FIG. 4C: carrying out OGD model processing for 4 h; FIG. 4D: OGD model treatment for 6h, FIG. 4E statistics of cell viability at various time points;
fig. 5 is a graph of the effect of melatonin-treated neural stem cells under hypoxic-ischemic conditions, fig. 5A: processing an OGD model for 2 h; FIG. 5B: carrying out OGD model processing for 4 h; FIG. 5C: carrying out OGD model processing for 6 h; FIGS. 5A, 5C are the single cell culture conditions of neural stem cells, FIGS. 5D, 5F, and 5E are the statistics of the cell survival rate after 2h, 4h, and 6h treatment, respectively, wherein each group is set with 0, 6.25, 12.5, 25, 50, and 100 μmol/L six groups of melatonin sites with different concentrations
FIG. 6 is a diagram showing the structure of the motor cortex of M1 and M2 in a map of the rat brain;
fig. 7 is a sample of rat brain injury model, fig. 7A: a schematic diagram of a skull injury model fracture bone window; FIG. 7B: after rat modeling, carrying out tissue fixation and whole brain drawing, and performing example of an injury area; FIG. 7C: fixing whole brain tissue, taking a longitudinal section of the brain tissue after material taking; 3mm of a scale;
FIG. 8 is the result of behavioral scoring using the mNSS scoring table for the improved rat neurological deficit score;
fig. 9 hematoxylin-eosin (H & E) staining of brain tissue sections of the injury area, wherein fig. 9A: obtaining a rat brain tissue, and then HE staining a microscopic image; FIG. 9B foci edge 200 μm cell count statistics;
fig. 10 is a cell repair effect and cell ratio statistics of damage area immunofluorescence staining detection assessment, wherein fig. 10A: co-staining Nestin with GFAP; FIG. 10B: nestin co-stained with Oligo 2; FIG. 10C: nestin was co-stained with MAP2, black in each histogram indicating the Nestin positive proportion, white indicating the GFAP/Oligo2/MAP2 positive proportion, scale 200 μm;
FIG. 11 is a graphical representation of the magnetic resonance T2 phase of rats at 7D post-implantation;
figure 12 is a post-transplant rat model behavioral scoring table using a nerve function impairment severity (NSS) scoring table, P < 0.05.
Detailed Description
The invention provides an application of melatonin in preparing a medicament for protecting nerve stem cells under an ischemic and anoxic condition. In the present invention, neural stem cells have the potential to differentiate into cells of a neural subset, which preferably includes glial cells and neurons; the glial cells preferably include astrocytes and oligodendrocytes. The neuroprotective manifestation is preferably that melatonin renders neural stem cells viable under ischemic and hypoxic conditions. The invention adopts cobalt chloride (CoCl) -containing material2) The EBSS sugar-free culture medium simulates an anoxic sugar-lacking condition of a lesion area after injury, the survival capability of neural stem cells cultured under the condition is greatly reduced, and after the neural stem cells under the anoxic sugar-lacking condition are treated by 25 mu mol/L melatonin, the survival quantity of the neural stem cells is increased, so that the survival capability of the neural stem cells is improved. The result shows that the melatonin has no toxic action on the neural stem cells under the conditions of ischemia and hypoxia, has reliable biological safety and also has better neuroprotective effect.
The invention provides a nerve protection deviceA graft material comprising melatonin and neural stem cells. In the transplantation material, the final concentration of the melatonin solution is preferably not less than 25 mu mol/L, more preferably 27-35 mu mol/L, and the concentration of the neural stem cells is preferably 105～107Per ml, more preferably 106And/ml. The preparation method of the transplantation material preferably prepares a neural stem cell suspension and a melatonin solution, wherein the solvent used by the melatonin solution preferably adopts a neural stem cell culture medium. And mixing the prepared neural stem cell suspension and the melatonin solution to obtain the inhibiting material.
The invention provides a 3D (three-dimensional) transplantation system with a craniocerebral injury repair function, which comprises a transplantation material and matrigel. The composition and content of the graft material are the same as those of the graft material with neuroprotective function, which are not described herein again. The volume ratio of the matrigel to the melatonin solution and the neural stem cell suspension in the transplantation material is preferably 0.8-1.2: 0.8-1.2: 0.8 to 1.2, most preferably 1:1: 1. The kind of the matrigel is not particularly limited in the present invention, and matrigel known in the art, for example, matrigel obtained by a conventional commercial purchase method, may be used. For illustrating the composition and implementation method of the 3D stereotactic implantation system of the present invention, Matrix Gel is used as an example in the examples of the present invention, but it should not be construed as limiting the present invention. The Matrix Gel can effectively help the attachment and differentiation of nerve cells, and meanwhile, the matrigel can also effectively play a role in supporting a 3D structure of a tissue cavity formed by injury and provide a three-dimensional environment for cell growth. The matrigel can be replaced by other tissue materials with good biocompatibility, such as hydrogel materials, hyaluronic acid materials, extracellular matrix-like materials and the like. The transplantation material and matrigel matrix gel are mixed, filled and transplanted to a focal zone of a rat craniocerebral injury model, the repairing condition of the injury zone is observed, and the immunohistochemical result shows that in an experimental group in which melatonin and stem cells are co-transplanted, the cell amount around the focal zone is obviously increased, and the edge form of the focal zone is more complete. The immunofluorescence result shows that in injured rat brain tissues co-transplanted with melatonin and neural stem cells, glial cell precursor cells are obviously increased, and the brain tissues can better provide structural support protection and nutrition functions and promote survival of cells in a focal zone and nerve function repair.
In view of the effect achieved by the graft material or the 3D stereotactic graft system in the recovery of nerve function, the use of the graft material or the 3D stereotactic graft system in the recovery of nerve function or the treatment of craniocerebral injury. The invention also provides application of the transplantation material or the 3D stereo-transplantation system in preparation of a medicine for recovering the nerve function. Meanwhile, in view of the biological performance of the transplantation material or the 3D stereo-transplantation system in repairing the craniocerebral injury of the rat, the invention provides the application of the transplantation material or the 3D stereo-transplantation system in preparing the medicine for treating the craniocerebral injury. The pharmaceutical formulation of the present invention is not particularly limited, and those known in the art may be used. The method for preparing the medicament of the present invention is preferably a method for preparing a medicament well known in the art.
The following will explain in detail a 3D stereografting material system with neuroprotective function and its application provided by the present invention with reference to the examples, but they should not be construed as limiting the scope of the present invention.
Example 1
1. Materials and reagents
Main instrument
Name and origin of laboratory Instrument company
Stereomicroscope CarlZeiss Ltd, Germany
Sartorius electronic balance thermo corporation, usa
Electric constant temperature water bath Box Yueku, Nanjing
OLYMPUSIX71 inverted phase contrast microscope OLYMPUS, Inc., Japan
Gold clock of ophthalmic surgical instruments, Shanghai
Microscopic surgical instruments Riwoder, Shenzhen
SJ-CJ1FDQ Single super clean bench Sujing, Suzhou
HeracelilCO 2 incubator Thermofeisher Co., USA
LSM780 laser confocal microscope CarlZeiss, Germany
ST40 Low temperature centrifuge Thermofisiher, USA
Millipore ultra pure Water plant Millipore Inc., Germany
5427R ultra high speed cryogenic centrifuge Eppendorf Ltd, Germany
Micropipettor, Darongxingchuang, Beijing
PH Meter Mi corporation, Shanghai
1.2 reagents and consumables
Name and origin of reagent/consumable company
25cm2Cell culture flasks NEST Corp, tin-free
75cm2Cell culture flasks NEST Corp, tin-free
15mm glass-bottom Petri dish NEST Corp, tin-free
60mm cell culture dish NEST Corp, tin-free
100mm cell culture dish NEST Corp, tin-free
100 μm cell sieve Corning, USA
DMEM/F12Gibco, USA
B27 additive Gibco, USA
Recombinant EGFP protech Co, USA
Mouse recombinant bFGFPEpotech, USA
Accutase cell digest Gibco, USA
0.01MPBS buffer, doctor Deg, Wuhan
0.25% pancreatic enzyme Gibco, USA
Gibco, Inc. of Fetal Bovine Serum (FBS), USA
GlutamaxGibco, USA
Polyornithine (PLO) Sigma, Germany
TritonX-100Sigma, Germany
4% paraformaldehyde Rod, Wuhan, Inc
5% BSA doctor Co., Wuhan, Inc
Immunofluorescence staining-anti-dilution liquid Biyuntian, Shanghai
Immunofluorescent staining of the second antibody dilution liquid, Biyuntian, Shanghai
Donkeyanti-rabbitIgG-CFL488Santa, Inc., USA
Donkeyanti-mouseigG-CFL555Santa, Inc., USA
Donkeyanti-rabbitIgG-CFL555Santa, USA
Donkeyanti-mouseigG-CFL488Santa, Inc., USA
AlexaFluor488conjugatedphalloidin
reagentsLife corporation, UK
DaPI staining fluid doctor Deg, Wuhan Ltd
Anti-fluorescence quencher Boshide, Wuhan, Inc
Nestin antibody SOX2 antibody Santa, Inc., Sanying, Inc., Wuhan
DCX antibody Sanying, Wuhan
Oligo2 antibody Sanying, Wuhan corporation
GFAP antibody Abcam Corp., USA
Melatonin Melatoninowder, Sigma, 98% (TLC), Germany
DMSO Biyunshi, Shanghai
Anhydrous ethanol Chongqing Chuantong chemical Co
EBSS、CoCl2。
2. Experimental methods
2.1 neural Stem cell isolation and culture-related procedures
2.1.1 Primary culture of neural Stem cells
(1) The frontal cortex tissue from which the pia mater and blood vessels had been removed was transferred to a 10ml sterile centrifuge tube using a 5ml pipette, excess DMEM-F12 was carefully aspirated, 3ml of 0.25% pancreatin (1 ml per 3 fetal mouse frontal cortex tissues, increased or decreased depending on the amount of fetal mouse brain tissue isolated) was added, digested for 12min in a 37 ℃ water bath, carefully placed to avoid contamination of the centrifuge tube orifice. The centrifugal tube is slightly shaken every other interval to avoid the tissue from being agglomerated and sinking to the bottom, so that the tissue is fully contacted with the pancreatin;
(2) after digestion was completed, the centrifuge tube was moved into a clean bench, pancreatin was carefully aspirated, and then 5ml of 10% FBS was added, and the FBS was carefully gently blown in the middle about 10 times to sufficiently contact the tissue mass, thereby terminating digestion. Standing for 5min, removing supernatant after tissue precipitation, repeating for 2 times to completely stop digestion of tissue mass;
(3) the cells were washed twice with 5ml of DMEM-F12 medium, and the remaining FBS was washed off. Removing the DMEM-F12 culture medium after the tissue mass is precipitated;
(4) 3.5ml of neural stem cell complete medium (DMEM/F12+ 2% B27+20ng/ml EGF +20ng/ml bFGF) was added to the centrifuge tube containing the tissue mass, and the tissue mass was gently pipetted 6 times with a 5ml pipette. After the tissue mass had settled, the supernatant suspension was aspirated and filtered through a 100 μm sieve. Repeating the washing and blowing filtration for 3 times;
(5) the filtered suspension is evenly and lightly blown and then averagely transferred to 3 culture bottles for culture. Adding 10ml of culture medium into each culture bottle;
(6) observing under optical microscope to obtain single cell as neural stem cell, and placing in HERAcelli CO25% CO at 37 ℃ in an incubator2Culturing under the condition;
(7) and (4) arranging and picking up experimental instruments and consumables, treating experimental waste and cleaning an ultra-clean bench.
2.1.2 neural Stem cell passages
The neural stem cells have the characteristic of stem cell proliferation, and when suspension ball-shaped cell masses with the diameters of about 100 mu m are formed after 2-3 days, passage is carried out.
(1) Sucking the cell culture suspension from the culture bottle into a 50ml sterile centrifuge tube, slightly flushing the wall of the culture bottle for several times, and collecting the neural stem cell balls remained on the wall of the culture bottle;
(2) collecting the cell culture suspension in a centrifuge tube, and centrifuging for 5min at 300 r/min;
(3) transferring the centrifuged supernatant into another 50ml sterile centrifuge tube, and precipitating to obtain the collected neural stem cells;
(4) adding 3ml of Accutase cell digestive juice (stem cell grade) into a sterile centrifuge tube in which neural stem cell sediment is stored, digesting at room temperature for 12min, and gently shaking midway to promote the cell balls to fully contact with the Accutase digestive juice;
(5) centrifuging the supernatant collected in the step (3) at 1500 rpm for 5min, collecting the centrifuged culture medium supernatant, and continuously using the culture medium supernatant as subculture of the neural stem cells;
(6) after the digestion of Accutase is finished, 3ml of recovery culture medium is added into the digestion solution to stop the digestion. Gently blowing and beating the neural stem cells for 20-30 times by using a 5ml pipette to fully disperse the neural stem cells, and then centrifuging the neural stem cells for 4min at a centrifugal speed of 300 g;
(7) centrifuging, removing supernatant, and collecting the precipitate as neural stem cell. Adding the neural stem cell complete culture medium into the sediment, and re-suspending the neural stem cell sediment. Then supplementing half of the culture medium recovered in the step (5), mixing, and inoculating to 75cm2Continuously placing into a culture flask at 37 deg.C and containing 5% CO2Suspension culture in an incubator. The neural stem cell single cell after the passage is the P1 generation cell, and the neural stem cell single cell after the passage is cultured is the P1 generation cell ball when growing into the neural stem cell ball.
2.2 neural Stem cell identification and migration differentiation experiment
2.2.1 adherent growth treatment of neural Stem cells
Because the neural stem cells grow in suspension in a normal state, the neural stem cells need to be subjected to adherence treatment in the experiment so as to facilitate subsequent experiments.
(1) Using sterilized ddH2O preparing poly-ornithine (PLO) solution of 10 mg/ml;
(2) placing the culture dish to be inoculated, the six-hole plate and the like into a super clean bench for ultraviolet sterilization for 30min for later use;
(3) adding 10mg/ml PLO solution into a culture dish and a six-hole plate to uniformly cover the bottom plane, and placing the culture dish and the six-hole plate in an incubator at 37 ℃ for coating for 15-30 min. And (4) recovering the residual PLO solution in the culture dish and the pore plate to a centrifugal tube after coating, and repeatedly using for 2-3 times.
2.2.2 identification and differentiation potential of neural Stem cells
To confirm that the isolated and cultured cells are neural stem cells, P2 generation neural stem cell spheres are inoculated into a 15mm glass bottom culture dish coated by PLO, and immunofluorescence staining is carried out on characteristic markers of the neural stem cells after three days of culture. In order to further verify the multidirectional differentiation potential of the neural stem cells, the complete neural stem cell culture medium in the neural stem cell ball which is cultured in a glass-bottom culture dish for three days is replaced by a neural stem cell differentiation culture medium (DMEM/F12+ 2% B27+ 1% glutamax), the neural stem cell ball is continuously cultured for three days to induce the differentiation, then immunofluorescence staining is carried out, and the differentiation markers are detected.
2.2.3 immunofluorescent staining
The immunofluorescence assay mainly aims at special protein molecular markers carried by different types of cells, combines protein molecules with designed fluorescent markers by using antibody reaction, and observes and analyzes by means of a fluorescence imaging technology. After the cell culture process of the previous experiment, the dryness of the neural stem cells is identified by immunofluorescence expression detection of neural stem cell specific markers Nestin and SOX2, and the neural stem cells are confirmed to be separated and cultured. After the differentiation medium is replaced to induce the differentiation potential, whether the neural stem cells have the multidirectional differentiation potential or not is shown by detecting the expression of specific molecular markers such as an astrocyte marker GFAP, an oligodendrocyte marker Oligo2, a neuroblast marker DCX and the like.
TABLE 1 marker antibody selection
The specific process of immunofluorescence is as follows:
(1) taking out neural stem cells (spheres) which are pre-inoculated in a PLO coated 15mm glass bottom culture medium at a set detection time point, sucking the culture medium in a culture dish, adding a proper amount of 0.01MPBS buffer solution according to the size of the culture dish, washing, and repeating for 2 times;
(2) after cleaning, sucking 0.01MPBS buffer solution in a culture dish, adding a proper amount of 4% paraformaldehyde to fix cells, and standing for 30min at room temperature;
(3) after the fixation is finished, 4% paraformaldehyde is completely absorbed, and a proper amount of 0.01MPBS is added for gentle shaking and washing for 3 times, wherein each time lasts for about 5 min;
(4) adding 0.3% TritonX-100 prepared by 0.01MPBS, standing at room temperature for 30min to increase permeability of cell membrane;
(5) removing 0.3% TritonX-100 membrane permeation solution by suction, adding 0.01MPBS, and washing for about 5min each time by gentle shaking;
(6) sucking residual 0.01MPBS buffer solution after washing, adding 200 ul 5% BSA, standing at room temperature, and blocking for 2 h;
(7) after blocking, 5% BSA was removed, and 200. mu.l primary antibodies were added at different concentrations, namely Nestin antibody (1: 100), Olig2 antibody (1: 200), DCX antibody (1: 200), and GFAP antibody (1: 1000), depending on the experimental groups and requirements. A negative control group was set, and 200. mu.l of the immunofluorescence-anti dilution used to dilute the antibody was added. The dishes were incubated overnight in an immunohistochemical wet box at 4 ℃ in a refrigerator.
(8) Taking out the culture dish, removing the primary incubation antibody by suction, and adding appropriate amount of 0.01MPBS buffer solution to clean for 3 times, each time for 5 min;
(9) absorbing 0.01MPBS buffer solution, adding 200 μ l of corresponding IgG fluorescent secondary antibody (1: 400) into different culture dishes according to the primary antibody source, and incubating at room temperature in the dark for 2 h;
(10) the fluorescent secondary antibody was aspirated and washed 3 times 5min each with 0.01 MPBS.
(11) When the cells need to be subjected to double-antibody co-labeling or triple-antibody co-labeling staining, such as SOX2 and Nestin double-labeling staining, after the incubation of the Nestin fluorescent secondary antibody is finished, the cells need to be washed by 0.01MPBS, 200 mu l of SOX2 antibody (1: 200) which is different from the Nestin primary antibody is added, and then the cells are placed in an immunohistochemical wet box at 4 ℃ for incubation overnight;
(12) absorbing the incubated primary antibody again, and adding 0.01MPBS buffer solution to wash for 3 times, each time for 5 min;
(13) after 0.01MPBS buffer solution is absorbed, 200 mu l of IgG fluorescent secondary antibody (1: 400) with different excitation wavelengths from the previously added fluorescent secondary antibody is added and incubated for 2h at room temperature in a dark place;
(14) absorbing the incubated fluorescent secondary antibody, and washing with 0.01MPBS buffer solution for 3 times, each time for 5 min;
(15) discarding 0.01M PBS used for washing, adding 200 μ l DAPI staining solution, placing in room temperature environment and incubating for 10min in dark place;
(16) absorbing DAPI staining solution, adding 0.01MPBS buffer solution, and washing for 5min each time for 3 times;
(17) after the washing is finished, adding a proper amount of 0.01M PBS buffer solution into the culture dish, dripping an anti-immunofluorescence quenching sealing agent to prevent fluorescence quenching, observing and taking a picture under a laser confocal microscope, and keeping the picture; if the observation and detection cannot be carried out in time, the culture dish should be stored in a refrigerator at 4 ℃ in a dark place.
(18) After observation and detection, the images are subjected to data analysis and processing by using Zen2011 software.
2.4 melatonin treatment of neural Stem cells in the OGD model
In order to explore the functional effect of melatonin on neural stem cells, CoCl was selected in vitro2+ EBSS chemical OGD model making method, set melatonin concentration gradient, test its protective effect on neural stem cells. The neural stem cells were previously seeded in a PLO-coated six-well plate. Observing that the neural stem cells have attached to the wall after about 2 hours, and replacing the complete culture medium of the neural stem cells with CoCl containing melatonin according to experimental grouping settings2+ EBSS mixed culture medium with melatonin concentration of 0, 6.25, 12.5, 25, 50, 100. mu. mol/L, and 5% CO at 37 deg.C2The cell culture box is used for culturing for 8 hours. After 8h, the cell culture plate was removed and the cell morphology was observed under an inverted phase contrast microscope.
3 results and analysis
3.1 isolated culture of neural Stem cell spheres
According to the procedures in the experimental method, neural stem cells were isolated and cultured in vitro in a neural stem cell complete medium in suspension. The optical morphology observation under the inverted phase contrast microscope can observe that the freshly extracted neural stem cells are high-brightness well-refracted small round cells (figure 1A). Extracting to obtain cellsStanding at 37 deg.C for 5% CO2After three days of standing culture in the cell culture box with the concentration, the neural stem cell balls formed by multi-cell aggregation can be obviously observed, the boundary is clear, and the cell aggregation state is good (figure 1B).
3.2 neural Stem cell identification
After the cultured cells are successfully separated in vitro, in order to further clearly verify that the cells are determined to be the neural stem cells, the cells are subjected to immunofluorescence staining identification by using molecular markers Nestin and SOX2 specific to the neural stem cells. By analyzing the results of immunofluorescent staining, the cultured cells were found to have a positive ratio of Nestin to the molecular marker SOX2 of 90% or more, and the isolated and cultured cells were fully proved to be neural stem cells.
3.3 differentiation potential of neural Stem cells
After the cells which are definitely separated and cultured are confirmed to be the neural stem cells, the neural stem cells need to be further verified to have the multidirectional differentiation potential of differentiating into neurons, oligodendrocytes and astrocytes. Therefore, the P2 generation neural stem cell ball is planted in a PLO coated confocal culture dish, the complete neural stem cell culture medium is replaced by a neural stem cell differentiation culture medium, and the differentiated cells are identified by using an immunofluorescence staining method after three days of culture. After analyzing the identification result, the neural stem cells cultured for three days can express a neuron precursor cell (neuroblast) specific marker DCX, an astrocyte specific marker GFAP and an oligodendrocyte marker Oligo 2. The isolated neural stem cells are confirmed to have a multipotentiality.
3.4 construction of model of oxygen sugar deprivation model (OGD)
In general craniocerebral injury, the damage of brain parenchyma caused by the injury often causes a series of pathological features such as bleeding, oxidative stress, neuroinflammatory reaction and the like, and influences the change of local microenvironment around a focal region. In various pathological environments with pathogenic damage, nerve function damage conditions which are caused by insufficient local blood and oxygen supply after simulated damage are selected, and an oxygen sugar deprivation (OGD) model is established at a cellular level. In this experiment, the neural stem cells were completely cultured in the mediumThe conversion was 100. mu. mol/L CoCl2The EBSS medium of (1). In the OGD model, the viability of neural stem cells is greatly reduced.
3.5 Effect of melatonin on neural Stem cell survival in the OGD model
Melatonin is more used as a rhythmic information hormone in the nervous system to regulate the consistency of the body and the light cycle of the environment. In multiple studies, melatonin is found to regulate the life process of neural stem cells by regulating mitochondrial functions, cell ion channels and other modes. Therefore, melatonin is added into an OGD (one-dimensional solution) model of the neural stem cells as a pre-conditioning condition, and whether the melatonin has a certain protective function under the condition that the OGD model damages the neural stem cells or not is tried to be researched. Under the same OGD model treatment conditions, different concentrations of melatonin treatments of 0, 6.25, 12.5, 25, 50 and 100 mu mol/L are set, and the survival state of the cells is observed and detected at each time point (figure 3). Research and observation show that after the OGD model is processed, the neural stem cells are all subjected to obvious apoptosis, the cells are rarely survived after the OGD model is processed for 6 hours, and most of the cells are dead to form a floating disorder state. (fig. 5C) comparative analysis revealed that the neural stem cells survived the most under 25 μmol/L melatonin treatment conditions in the six sets of melatonin dose treatments, indicating that melatonin had a better level of protection against neural stem cell apoptosis under OGD model treatment conditions.
Example 2
1. Materials and reagents
1.1 Main Material
220-250 g male SD rats purchased from the Experimental animals center of army and military medical university.
1.2 Main instruments
Name and origin of laboratory Instrument company
OPMIpico desktop surgical microscope Zeiss Corp Germany
Microscopic surgical instruments, medical diffuse medical instruments and Shenzhen
Haier company of refrigerator, Qingdao
Spring light medical beauty treatment apparatus of Bipolar micro-surgical electrocoagulator, Wuhan, Inc
Gold clock of ophthalmic surgical instruments, Shanghai
Small animal stereotaxic instrument, Shenzhen
Dental drill
1.3 reagents and consumables
Name and origin of reagent/consumable company
5% Chloral hydrate solution Cron Chemicals, Inc., Chengdu City
4% paraformaldehyde solution, Colon Chemicals, Inc., Chengdu City
Medical Iodophor-Shirte chemical reagent, Jinan
3% hydrogen peroxide (H2O2) solution Schalt chemical, Inc., of Jinan
Chongqing pharmaceutical products of southwestern physiological saline
2 method of experiment
2.1 rat craniocerebral injury model preparation
Based on the traditional impact wound model making method in the past, the positioning method is specifically positioned in the moving cortical areas of rats M1 and M2, the positioning method is based on a third version rat brain stereotaxic map (original George Paxin, Charles Watson), rat bone windows are further broken, cortical tissues are removed, and tissue deformities and pathological environments formed after craniocerebral injury are simulated, and the specific operation steps are as follows:
(1) weighing a purchased rat experiment table, and injecting 5% chloral hydrate solution into the abdominal cavity according to the proportion of 50mg/kg to anaesthetize the rat;
(2) after the rat is anesthetized, the hair on the top of the skull is removed and fixed on an in vitro positioning instrument of the rat;
(3) the epidermis of the rat head was wiped with iodophors, and the parietal skin was then cut forward from the hindbrain along the coronal midline between the two eyes, exposing the skull, and then periosteum was removed using a 3% hydrogen peroxide (H2O2) solution;
(4) in the bregma position according to the predetermined skin area with the movement function of rats M1 and M2, a boundary line is drawn in the area 1-4 mm in front of the bregma and 1-4 mm on the right by using a pair of microscratches, and the range of a bone window is encircled;
(5) the skull is carefully abraded along the boundary line using a dental drill according to a predetermined bone window range, and the bone fragments are removed. When the bone window is opened, the normal saline is timely injected around the drill bit, so that the brain tissue thermal injury caused by local high temperature of the drill bit is avoided, and the dura mater is not worn out;
(6) in the range of the bone window, the dura mater is carefully cut off by using a microscopic instrument, so that the internal blood vessel is prevented from being damaged;
(7) after cutting off dura mater, clamping and sintering a relatively obvious blood vessel by using bipolar coagulation, then cutting the blood vessel along the edge of a bone window for 3mm deep to separate a middle exposed tissue from surrounding brain tissues, and then clamping the brain tissues in a middle injury area by using forceps to form an injury cavity;
(8) clamping expected injured brain tissue, performing hemostasis on the tissue in the injured area by using bipolar electrocoagulation, and then suturing the skin in sequence;
(9) the rats were placed on an incubator for resuscitation and returned to the rearing chamber after reviving for seven days.
2.2 behavioral Scoring
Model rats were evaluated for neurological function by behavioral scoring on the seventh day after model creation. We used a modified rat neurological deficit score, mNSS, as specified in table 2 below:
TABLE 2 modified rat neurological deficit score mNSS Table
Exercise test
6
Tail lifting test
3
Flexion of forelimbs
1
Hind limb flexion
1
Head deviating from vertical axis within 30s>10°
1
Rats were placed on the floor (normal 0; maximum 3)
3
Normal walking
0
Can not walk in straight line
1
Side-rotating ring for paraplegia
2
Incline and fall to the side of the paresis
3
Sensory test
2
Placement test (visual and tactile testing)
1
Proprioceptive test (deep feeling, pressing mouse paw to table edge to stimulate limb muscle)
1
Balance beam test (Normal)The value is 0; maximum value 6)
6
Stabilize the balance posture
0
Edge of the grab balance beam
1
Hug the balance beam tightly, one limb falls from the balance beam
2
Hug the balance beam, and the two limbs drop from the balance beam or rotate on the balance beam: (>60 seconds)
3
Attempt to balance on the balance beam but fall (>40 seconds)
4
Attempt to balance on the balance beam but fall (>20 seconds)
5
Dropping; no attempt is made to balance on the balance beam (<20 seconds)
6
Loss of reflexes and abnormal movements
4
Auricle reflex (shaking head when contacting external auditory canal)
1
Corneal reflex (blink when touching the cornea with cotton silk)
1
Panic reflex (moving response to the noise of quick-bouncing hardboard)
1
Epilepsy, myoclonus, dystonia
1
Highest score 18
2.3 fixed sampling of rat brain tissue
The rats after seven days of feeding were subjected to tissue fixation using a 4% paraformaldehyde solution, and then brain tissues of the rats were dissected out and subjected to evaluation comparison of the injured area.
(1) Weighing rats, injecting 5% chloral hydrate solution into abdominal cavity according to the proportion of 50mg/kg for anesthesia, placing the rats into a tray, and preparing a 20ml syringe, normal saline and 4% paraformaldehyde which is cooled in advance for later use;
(2) after anesthetizing the rat, the chest was cut open and the chest ribs were fixed up with clips, exposing the rat heart to the visual field;
(3) the left apex of the heart is punctured by a perfusion needle filled with normal saline, then the upper part of the right auricle is cut, the normal saline is slowly injected, the blood is discharged through an internal circulation system until clear liquid flows out from the right auricle and the blood is emptied;
(4) the rat was replaced with a syringe containing a pre-cooled 4% paraformaldehyde solution and pushed into the body of the rat slowly as well, so that the 4% paraformaldehyde solution fixed the tissue via the circulatory system, and twitching of the rat limbs and tail was observed. After the body of the rat is stiff, fixing is finished;
(5) after fixation, cutting off the head along the neck, cutting off the head skin from the hindbrain along the coronal midline, removing the skull, taking out the brain tissue, and then placing into 4% paraformaldehyde solution for dark preservation;
(6) after brain tissue is fixed in 4% paraformaldehyde solution for one day, the brain tissue is taken out and put into 30% paraformaldehyde sucrose solution (prepared by adding 30g of sucrose into 4% paraformaldehyde per 100 ml), and the 30% paraformaldehyde sucrose solution is replaced for three days after dehydration until the brain tissue of the rat floats on the upper layer of the solution.
3 results and analysis
3.1 rat craniocerebral injury model
In order to better simulate the function damage after craniocerebral injury, the brain stereotaxic map of a third edition of rats aims at the M1 and M2 motor cortex to carry out injury (as shown in figure 6), and the motor function damage condition and the subsequent repair and recovery effect are evaluated.
After positioning according to the atlas, accurately positioning the focal zone of craniocerebral injury in M1 and M2 motor cortex areas, and then cutting off the brain tissue in the area in a manner of damaging the cortex area through craniotomy to form a 3 multiplied by 3mm3The size of the lesion cavity. In order to verify the consistency of the control of the injury range in the molding process, the tissue is fixedly taken from the rat seven days after the molding, the whole brain tissue injured by the craniocerebral injury model is completely taken out, and the size of the cavity of the injured focus is observed and measured. As can be seen in fig. 7, the lesion area of each rat was measured to be consistent with the pre-planned M1, M2 motor cortex lesions. After the lesion cavities of each craniocerebral injury rat model are measured and compared, the sizes of the cavities caused by each model rat are basically consistent without obvious difference, and the deviation is within an allowable error range. Analysis in conjunction with rat brain maps also suggests that there are no unmeasurable differences in functional compartmentalization of the lesion area. Therefore, the craniocerebral injury model has consistency among groups in morphological and functional partitions, and the feasibility of the novel brain injury model developed by the research is proved.
3.2 evaluation of craniocerebral injury models
After the consistency among the craniocerebral injury model groups is verified, a manufacturing method for the model process is formulated, and the problem of model manufacturing is solved. On the basis of model making, in order to verify that the model after craniocerebral injury has definite functional injury on nerve functional behaviors, each experimental rat is independently scored in a behavioural way seven days after model making. The scoring mode selects a nerve injury severity scoring (NSS) table, and the nerve function injury condition after craniocerebral injury is statistically analyzed according to the scoring of each experimental rat, wherein the statistical data is shown in figure 8(Sham group is a control Sham operation group, and TBI is a craniocerebral injury group). Statistical data show that the craniocerebral injury model rats have obvious nerve function injury compared with the control group.
Example 3
1 materials and reagents
1.1 Main Material
220-250 g of male SD rats purchased from the experimental animal center of army and military medical university;
isolating the cultured neural stem cells in vitro.
1.2 Main instruments
Name and origin of laboratory Instrument company
Stereomicroscope CarlZeiss Ltd, Germany
Sartorius electronic balance thermo corporation, usa
Electric constant temperature water bath Box Yueku, Nanjing
OLYMPUSIX71 inverted phase contrast microscope OLYMPUS, Inc., Japan
Gold clock of ophthalmic surgical instruments, Shanghai
Microscopic surgical instruments Riwoder, Shenzhen
SJ-CJ1FDQ Single super clean bench Sujing, Suzhou
HERAcelli CO2Incubator Thermofoisher Inc., USA
LSM780 laser confocal microscope CarlZeiss, Germany
ST40 Low temperature centrifuge Thermofisiher, USA
Millipore ultra pure Water plant Millipore Inc., Germany
5427R ultra high speed cryogenic centrifuge Eppendorf Ltd, Germany
Micropipettor, Darongxingchuang, Beijing
PH Meter Mi corporation, Shanghai
OPMIpico desktop surgical microscope Zeiss Corp Germany
Microscopic surgical instruments, medical diffuse medical instruments and Shenzhen
Haier company of refrigerator, Qingdao
Spring light medical beauty treatment apparatus of Bipolar micro-surgical electrocoagulator, Wuhan, Inc
Gold clock of ophthalmic surgical instruments, Shanghai
Small animal stereotaxic instrument, Shenzhen
Dental drill
1.3 reagents and consumables
Name and origin of reagent/consumable company
25cm2Cell culture flasks NEST Corp, tin-free
75cm2Cell culture flasks NEST Corp, tin-free
15mm glass-bottom Petri dish NEST Corp, tin-free
60mm cell culture dish NEST Corp, tin-free
100mm cell culture dish NEST Corp, tin-free
100 μm cell sieve Corning, USA
DMEM/F12Gibco, USA
B27 additive Gibco, USA
Recombinant EGFP protech Co, USA
Mouse recombinant bFGFPEpotech, USA
Accutase cell digest Gibco, USA
0.01MPBS buffer, doctor Deg, Wuhan
0.25% pancreatic enzyme Gibco, USA
Gibco, Inc. of Fetal Bovine Serum (FBS), USA
GlutamaxGibco, USA
Polyornithine (PLO) Sigma, Germany
TritonX-100Sigma, Germany
4% paraformaldehyde Rod, Wuhan, Inc
5% BSA doctor Co., Wuhan, Inc
Immunofluorescence staining-anti-dilution liquid Biyuntian, Shanghai
Immunofluorescent staining of the second antibody dilution liquid, Biyuntian, Shanghai
Donkeyanti-rabbitIgG-CFL488Santa, Inc., USA
Donkeyanti-mouseigG-CFL555Santa, Inc., USA
Donkeyanti-rabbitIgG-CFL555Santa, USA
Donkeyanti-mouseigG-CFL488Santa, Inc., USA
AlexaFluor488conjugatedphalloidin
reagentsLife corporation, UK
DaPI staining fluid doctor Deg, Wuhan Ltd
Anti-fluorescence quencher Boshide, Wuhan, Inc
Nestin antibody SOX2 antibody Santa, Inc., Sanying, Inc., Wuhan
DCX antibody Sanying, Wuhan
Oligo2 antibody Sanying, Wuhan corporation
GFAP antibody Abcam Corp., USA
Melatonin Melatoninowder, Sigma, 98% (TLC), Germany
DMSO Biyunshi, Shanghai
Anhydrous ethanol Chongqing Chuantong chemical Co
EBSS
CoCl2
5% Chloral hydrate solution Cron Chemicals, Inc., Chengdu City
4% paraformaldehyde solution, Colon Chemicals, Inc., Chengdu City
Medical Iodophor-Shirte chemical reagent, Jinan
3% of hydrogen peroxide (H)2O2) Shiertai chemical reagent, Jinan, solution
Chongqing pharmaceutical products of southwestern physiological saline
2 method of experiment
2.1 graft Material mixing and grouping
The specific matching method of the material transplantation group is as follows:
the preparation before the experiment is carried out, each group of 45 mu l system is estimated to use 10 mu l/cell, and the cell quantity of the neural stem cell suspension is about 106Per ml
1. Matrigel group: 30. mu.l of neural stem cell complete medium + 15. mu.l of MTX matrigel
2. Neural stem cell suspension group: 30. mu.l of neural stem cell complete medium + 15. mu.l of neural stem cell suspension
3. Neural stem cell suspension + MTX matrigel group: 15 μ l of neural stem cell complete medium +15 μ l of MTX matrigel +15 μ l of neural stem cell suspension
4. The neural stem cell melatonin matrigel transplantation group comprises: for co-culture transplantation of neural stem cells and melatonin, 15 mu l of 75 mu M melatonin solution (prepared by a complete neural stem cell culture medium) +15 mu l of MTX matrigel +15 mu l of neural stem cell suspension, on the basis of common transplantation, MatrixGel matrigel is mixed as a 3D structural support material, and the following groups are set according to a single variable principle:
group of MatrixGel Matrigel (MTX); ② neural stem cell suspension (NSC) group; ③ the group of the neural stem cell suspension and matrigel (NSC + MTX); and fourthly, the group of the neural stem cells, Matrix Gel and melatonin (NSC + MTX + Mel). And in order to maintain the consistency of cells among each group, counting the cells of each group before mixing to ensure that the number of the neural stem cells is maintained at 1 multiplied by 10 when each brain injury model rat is transplanted5And (4) cells. In addition, in order to avoid influencing the repairing effect of cells in a focus area due to the strength difference of colloid filling after transplantation caused by the concentration difference of the matrigel, the 2 nd group is liquid transplantation, and the content ratio of the matrigel transplantation of the other groups is 1/3 of the total volume.
2.2 immunohistochemical hematoxylin-eosin (H & E) staining
(1) Weighing rats, injecting 5% chloral hydrate solution into abdominal cavity according to the proportion of 50mg/kg for anesthesia, placing the rats into a tray, and preparing a 20ml syringe, normal saline and 4% paraformaldehyde which is cooled in advance for later use; after anesthetizing the rat, the chest was cut open and the chest ribs were fixed up with clips, exposing the rat heart to the field of view; puncturing a perfusion needle filled with normal saline into the left apex of the heart, then shearing the upper part of the right auricle, slowly injecting the normal saline, discharging blood through an internal circulation system until clarified liquid flows out of the right auricle and the blood is emptied; the rat was replaced with a syringe containing a pre-cooled 4% paraformaldehyde solution and pushed into the body of the rat slowly as well, so that the 4% paraformaldehyde solution fixed the tissue via the circulatory system, and twitching of the rat limbs and tail was observed during the process. After the body of the rat is stiff, fixing is finished; after fixation, cutting off the head along the neck, cutting off the head skin from the hindbrain along the coronal midline, stripping off the skull, taking out the brain tissue, and then putting into 4% paraformaldehyde solution for dark preservation; after brain tissue is fixed in 4% paraformaldehyde solution for one day, taking out and putting into 30% paraformaldehyde sucrose solution (prepared by adding 30g of sucrose into 4% paraformaldehyde per 100 ml), dehydrating for three days, replacing 30% paraformaldehyde sucrose solution until the brain tissue of a rat floats on the upper layer of the solution, rinsing with 0.01MPBS buffer solution after fixation to remove redundant fixative, and transferring the brain tissue into 70% ethanol for storage and standby;
(2) the gradient alcohol dehydration method comprises sequentially placing brain tissue into alcohol solution with the following concentration for dehydration treatment, wherein the dehydration treatment comprises 70%, 80%, 90% and anhydrous ethanol, and each step is about ten minutes. Then sequentially using 50% xylene ethanol solution, xylene and second xylene, and carrying out gradient treatment for 12-15 minutes each time, wherein the 50% xylene ethanol mixed solution needs to be prepared for use in situ;
(3) placing the tissue in a small beaker which is provided with melting wax and has a volume of 100ml and corresponds to the tissue, carrying out wax penetration in a soft wax beaker for 1 hour at the constant temperature of 60 ℃, and transferring the tissue to a hard wax beaker for wax penetration for 1 hour;
(4) embedding paraffin, embedding the tissue with molten liquid wax at 60 deg.c, fixing the tissue in advance mark and storing at 4 deg.c;
(5) slicing the tissue, fixing and mounting the tissue on a slicer, and shaking a handwheel of the slicer at a constant speed to obtain 5-micron slices;
(6) quickly spreading the cut wax tape facing to the blade surface in warm water at 37 ℃ for 1-2 minutes by using a pair of tweezers, completely flattening the wax tape, fishing out the wax tape after the wax tape is completely flattened, slightly fishing out the wax tape by using a glass slide coated with polylysine, and preferably positioning the wax tape in the center of the glass slide to prevent tissues from being abraded by post-treatment;
(7) baking the whole brain paraffin slices in a 65 ℃ oven for half an hour, or baking the whole brain paraffin slices in a 38 ℃ baking machine for one night to firmly adhere the paraffin strips;
(8) dewaxing, namely sequentially dewaxing by using first xylene and dewaxing by using second xylene for 15 minutes respectively;
(9) and (4) gradient hydration. The slides were immersed in 100% ethanol 2 times for 5 minutes each, followed by 5 minutes each in a solution of 90%, 80%, 70% ethanol. Rinsing the slices in double distilled water for 5 minutes and then dyeing;
(10) and (3) hematoxylin staining: after staining the tissue sections in hematoxylin stain for 5 minutes, the sections were rinsed in tap water for an appropriate time until the color of the sections turned blue. While observing, the slices were differentiated in 1% hydrochloric acid ethanol differentiation solution, and when the slices became red, the differentiation was stopped, and the slices were washed carefully with tap water to turn blue
(11) Eosin staining: the sections were stained in eosin for 2 seconds, followed by 75% ethanol, 85% ethanol, 95% ethanol, 100% ethanol II, 100% ethanol I, xylene II, and xylene I for 5 seconds, respectively, and washed to remove excess loose color. The xylene is transparent twice continuously, and each xylene is baked in an oven at 65 ℃ for 5 minutes
(12) Sealing, namely dripping a drop of neutral gum on one side of the slice by using a rubber head dropper, slightly pressing the cover glass sheet, and applying uniform force without sliding the cover glass sheet on the glass slide and avoiding bubbles;
(13) microscopic observation and photographing: and observing and shooting after the to-be-sliced sheets are dried.
2.3 immunofluorescence staining of tissue sections
2.3.1 frozen sections
(1) The rat brain tissue was removed and dehydrated according to the experimental method in 2.2 (1);
(2) taking out the dehydrated brain tissue, cutting off cerebellum, and flatly placing the brain tissue on a slice freezing table;
(3) completely embedding brain tissue on a freezing table layer by using an OCT embedding medium, and putting the brain tissue on a freezing slicer for freezing treatment to ensure that the embedding medium is completely solidified and is adhered to the freezing table;
(4) setting the slice thickness to be 16 mu m, placing the embedded brain tissue on a slice rack, and rotating a handle to sequentially slice;
(5) after the target area is sliced, the slices are taken by precooled tweezers and placed in an antifreeze solution (prepared by 30ml of ethylene glycol, 30g of cane sugar and 40ml of 0.1M PBS), and the slices are placed at-20 ℃ for storage after marking.
2.3.2 immunofluorescence staining of tissue sections
(1) Taking out the preserved slices, placing the slices in a 24-hole plate containing 0.01M PBS, cleaning for 3 times, 5 minutes each time, completely sucking the PBS in the hole plate after cleaning, and adding a proper amount of 0.3% Triton-X100 membrane for 30 minutes;
(2) after the membrane penetration, removing Triton-X100 by suction, and cleaning the specimen by using 0.01MPBS for 5 minutes each time for 3 times;
(3) after washing, absorbing PBS, adding a proper amount of 5% BSA, and sealing for 2h at room temperature;
(4) after the blocking is finished, absorbing 5% BSA blocking solution in the pore plate, adding a proper amount of Nestin immunofluorescence primary antibody (1: 100) into each pore, covering the pore plate, marking, placing the well-covered pore plate in an immunohistochemical wet box, and incubating in a refrigerator at 4 ℃ overnight;
(5) after incubation was complete, the well plate was removed, the primary antibody was recovered by aspiration, and then washed 3 times with 0.01MPBS gentle shaking for 5 minutes each time;
(6) completely sucking PBS in the pore plate, adding a proper corresponding immunofluorescence secondary antibody into the pore plate according to the source of the antibody, and incubating for 2 hours at room temperature in a dark place;
(7) the immunofluorescence secondary antibody was aspirated off, and the well plates were washed 3 times with 0.01M PBS for 5 minutes each time;
(8) when the cells need to be stained by a double antibody co-labeling or a triple antibody co-labeling, for example, GFAP, MAP2, Oligo2 and Nestin are stained by a double antibody co-labeling, after the incubation of the Nestin fluorescent secondary antibody is completed, the cells need to be washed by 0.01MPBS, 200 μ l of GFAP antibody (1: 1000), MAP2 antibody (1: 200) and Oligo2 antibody (1: 200) from different sources from the Nestin primary antibody are added, and then the cells are placed in an immunohistochemical wet box at 4 ℃ for overnight incubation;
(9) the incubated primary antibody was again aspirated off, washed 3 times with 0.01M PBS buffer, 5 minutes each time;
(10) after 0.01MPBS buffer solution is aspirated, 200. mu.l of IgG fluorescent secondary antibody (1: 400) with different excitation wavelengths from the previously added fluorescent secondary antibody is added and incubated for 2 hours at room temperature in a dark place;
(11) absorbing the incubated fluorescent secondary antibody, and washing with 0.01M PBS buffer solution for 3 times, 5 minutes each time;
(12) discarding 0.01MPBS used for cleaning, adding 200 mul of DAPI staining solution, and placing in a room temperature environment for incubation for 10 minutes in a dark place;
(13) absorbing DAPI staining solution, adding 0.01M PBS buffer solution, and washing for 3 times, each time for 5 min;
(14) after the washing is finished, adding a proper amount of 0.01M PBS buffer solution into the culture dish, dripping an anti-immunofluorescence quenching sealing agent to prevent fluorescence quenching, observing and taking a picture under a laser confocal microscope, and keeping the picture; if the observation and detection cannot be carried out in time, the culture dish should be stored in a refrigerator at 4 ℃ in a dark place.
(15) After observation and detection, the images are subjected to data analysis and processing by using Zen2011 software.
3 results and analysis
3.1 HE staining of lesion area
In order to determine the cell repair condition of a model rat in each experimental grouping around a focal zone after melatonin and neural stem cells are transplanted in a rat craniocerebral injury model together, the model rat is perfused and taken, brain tissues are sequentially dehydrated, and paraffin sections are carried out. And (3) after paraffin sectioning, performing hematoxylin-eosin staining according to immunohistochemical requirements, marking the cell morphology around the focal zone, counting the number of cells within 200 mu m of the edge of the focal zone, and judging the repairing difference. According to the staining of immunohistochemical sections, the experimental model with Matrix Gel Matrix added is that cells around a lesion area of a rat are dense and have obvious connection and fusion phenomena with materials. As can be clearly found in the counting analysis of the cells within the range of 200 μm around the focal region, the numbers of the peripheral cells of the neural stem cell matrigel binding group and the neural stem cell + matrigel + melatonin group are obviously increased compared with the matrigel group and the neural stem cell suspension group. In the experimental group for transplanting the neural stem cells, the matrigel and the melatonin together, the number of cells around the focal region is obviously increased compared with that of the neural stem cell transplanting group without melatonin treatment, and basically, the fact that the melatonin can effectively promote the growth of the neural stem cells in the treatment of cell transplantation, protect the neural stem cells from surviving in a focal cavity after the craniocerebral injury of a rat and increase the survival rate of the cells in the focal region can be inferred.
3.2 immunofluorescence detection of cell transplantation repair levels
In order to further verify the nerve function repair effect of melatonin and neural stem cells after co-transplantation and explore the differentiation fate of the neural stem cells after transplantation to the lesion focal region, after taking whole brain tissues, frozen sections are carried out in the range of the lesion region, and after the sections are respectively stained by immunofluorescence using a neuron marker MAP2, an astrocyte marker GFAP, oligodendrocytes Oligo2 and a neural stem cell marker Nestin, the cell types around the lesion region are detected, and the function damage repair strategy of the neural stem cells under pathological conditions is presumed.
Analysis of immunofluorescent staining results revealed that Nestin + positive cells were significantly increased around the lesion focus and consistent with immunohistochemical results. Staining analysis of GFAP, Oligo2 and MAP2 differentiation markers revealed that there was no significant difference in staining for Oligo2 in the four transplanted groups (FIG. 12), and little or no differentiation of neural stem cells into Oligo 2-labeled oligodendrocytes. In the NSC + MTX transplantation group, the GFAP + positive rate was higher than that in the other groups. After the Mel treatment, the positive expression of MAP2 is increased, and the differentiation of the neural stem cells to the neurons is increased. Therefore, the transplantation effect of the neural stem cells is obviously improved after the matrigel is added in the lesion area of the normal injury model. Under conventional pathological conditions, the spontaneous differentiation fate of the neural stem cells is to differentiate to astrocytes around a focal region, and the differentiation into astrocytes plays a role in integrating damaged neural networks, providing a nutritional basis, regulating and controlling a damaged environment and improving so as to achieve the effect of repairing damaged neural functions. After melatonin is added, the differentiation fate of the neural stem cells develops towards the direction of the neurons, the absolute number of the neurons around a focal region is increased, a damaged regeneration neural network is constructed, and the neural function recovery effect is improved more directly.
3.3MRI magnetic resonance technique detection
After the rat craniocerebral injury model is made and the neural stem cell transplantation filling is carried out, the experimental rat is subjected to the small animal magnetic resonance detection on the seventh day. And the condition of the lesion in the lesion area is visually judged and evaluated by a magnetic resonance detection technology. In the magnetic resonance T2 phase, the highlighted reaction region is the focal region in the craniocerebral lesion. The magnetic resonance image can observe that the size of a highlight signal area in the magnetic resonance T2 phase of the NSC + MTX + Mel group is smaller than that of the MTX group, the NSC group and the NSC + MTX group, which shows that in a model rat which is subjected to a cell transplantation experiment and is seven days later, the lesion area of an experimental rat with the NSC + MTX + Mel group is better in recovery and has a better repairing effect.
3.4 behavioral Scoring of neurological function
In order to evaluate the difference in the effect of recovering neural function between groups in the neural stem cell transplantation experiment performed on the rat brain injury model, the behavioural analysis of the rat model was evaluated using a neural function injury severity (NSS) scale. Data analysis shows that the behavioral score of the rat transplanted and filled into the rat craniocerebral injury model together with melatonin and neural stem cells is obviously reduced compared with other experimental groups, and the nerve function recovery effect of the rat group after injury is superior to that of rats of other three groups of models. The matrix glue is proved to have the effects of supporting the tissue structure and promoting the recovery of the nerve function in the repair process.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.