Stem cell gel for nerve repair and preparation method and application thereof

文档序号：121107 发布日期：2021-10-22 浏览：33次中文

阅读说明：本技术 一种可用于神经修复的干细胞凝胶及其制备方法和应用 (Stem cell gel for nerve repair and preparation method and application thereof ) 是由曹毓琳滕睿頔程世翔白志惠于 2021-06-18 设计创作，主要内容包括：本发明提供了一种可用于神经修复的干细胞凝胶的制备方法,该制备方法具体包括以下步骤：(1)合成质粒序列：将G-CSF基因序列与2A串联合成G-CSF-2A片段,再与SDF-1基因序列串联合成G-CSF-2A-SDF-1片段,再将G-CSF-2A-SDF-1片段克隆到pRRLSIN.cPPT病毒表达载体上,即得质粒序列；(2)构建病毒：将质粒序列与psPAX2和PMD2.G采取三质粒包装系统进行病毒包装,即得携带表达G-CSF、SDF-1蛋白的慢病毒；(3)感染细胞：将步骤(2)制得的慢病毒加入P2代MSC细胞中进行感染,即得表达G-CSF和SDF-1的间充质细胞；(4)制备凝胶材料,将表达G-CSF和SDF-1的间充质细胞重悬于海藻酸钠水溶液中,再将重悬有细胞的海藻酸钠水溶液与氯化钙水溶液混合均匀,即得；该干细胞凝胶对神经损伤有很好的治疗效果。(The invention provides a preparation method of stem cell gel for nerve repair, which specifically comprises the following steps: (1) plasmid sequence synthesis: connecting the G-CSF gene sequence with 2A in series to synthesize a G-CSF-2A fragment, connecting the G-CSF-2A-SDF-1 fragment with the SDF-1 gene sequence in series to synthesize a G-CSF-2A-SDF-1 fragment, and cloning the G-CSF-2A-SDF-1 fragment to a pRRLSIN. cPPT virus expression vector to obtain a plasmid sequence; (2) constructing a virus: carrying out virus packaging on the plasmid sequence, psPAX2 and PMD2.G by adopting a three-plasmid packaging system to obtain lentivirus carrying expression G-CSF and SDF-1 proteins; (3) infecting the cells: adding the lentivirus prepared in the step (2) into MSC (mesenchymal stem cell) of P2 generation for infection to obtain mesenchymal cells for expressing G-CSF (granulocyte-colony stimulating factor) and SDF-1; (4) preparing a gel material, namely resuspending the mesenchymal cells expressing G-CSF and SDF-1 in a sodium alginate aqueous solution, and uniformly mixing the sodium alginate aqueous solution with the resuspended cells with a calcium chloride aqueous solution to obtain the gel material; the stem cell gel has good therapeutic effect on nerve injury.)

1. A preparation method of stem cell gel for nerve repair is characterized by comprising the following steps:

(1) plasmid sequence synthesis: connecting the G-CSF gene sequence with 2A in series to synthesize a G-CSF-2A fragment, connecting the G-CSF-2A-SDF-1 fragment with the SDF-1 gene sequence in series to synthesize a G-CSF-2A-SDF-1 fragment, and cloning the G-CSF-2A-SDF-1 fragment to a pRRLSIN. cPPT virus expression vector to obtain a plasmid sequence;

(2) constructing a virus: adopting a three-plasmid packaging system to package the plasmid sequence, psPAX2 and PMD2.G with lentivirus, and using HEK-293T cells as packaging cells of the lentivirus to obtain the lentivirus carrying and expressing G-CSF and SDF-1 proteins;

(3) infecting the cells: adding the lentivirus carrying expressed G-CSF and SDF-1 proteins prepared in the step (2) into a P2 generation mesenchymal stem cell for infection to obtain a mesenchymal cell expressing G-CSF and SDF-1;

(4) preparing a gel material: and (3) resuspending the mesenchymal cells expressing G-CSF and SDF-1 in a sodium alginate aqueous solution to prepare a sodium alginate aqueous solution with the resuspended cells, and uniformly mixing the sodium alginate aqueous solution with the resuspended cells and a calcium chloride aqueous solution to obtain the stem cell gel.

2. The method of claim 1, wherein the G-CSF-2A-SDF-1 fragment is cloned into pRRLSIN. cPPT virus expression vector at NheI and XbaI sites in step (1).

3. The method for preparing a stem cell gel useful for nerve repair according to claim 5, wherein the plasmid sequence in step (1) has the following structure:

pLenti-CMV-G-CSF-2A-SDF-1-EGFP-3FLAG-PGK-Puro。

4. the method for preparing a stem cell gel useful for nerve repair according to claim 1, wherein the mass ratio of the plasmid sequence to the psPAX2 and pmd2.g in the system in step (2) is as follows: plasmid sequence psPAX2 pmd2.g ═ 4:2-5: 1-3.

5. The method for preparing a stem cell gel useful for nerve repair according to claim 1, wherein the infected cells in the step (3) are screened by puromycin 48 hours after infection.

6. The method for preparing a stem cell gel useful for nerve repair according to claim 1, wherein the concentration of the sodium alginate aqueous solution is 0.1g/L and the concentration of the calcium chloride aqueous solution is 0.1g/L in the preparation of the gel material in the step (4).

7. The method for preparing a stem cell gel useful for nerve repair according to claim 6, wherein the gel material prepared in the step (4) is resuspended in 1X 10 per 10ml of the aqueous solution of sodium alginate⁸And (4) cells.

8. The method for preparing a stem cell gel useful for nerve repair according to claim 7, wherein in step (4), the aqueous calcium chloride solution is mixed with the aqueous sodium alginate solution in which the cells are resuspended in an equal volume.

9. A stem cell gel useful for nerve repair, prepared according to the method of any one of claims 1 to 8.

10. Use of the stem cell gel useful for nerve repair of claim 9 in a medicament for treating nerve injury.

Technical Field

The invention belongs to the technical field of cells, and particularly relates to a stem cell gel for nerve repair, and a preparation method and application thereof.

Background

The treatment of nerve injury is still one of the worldwide difficult diseases, and currently, the treatment of nerve injury in our country is difficult to completely cure, and the nerve injury is a non-renewable disease in medicine, that is, when cells in nerves are damaged, the cells are difficult to regenerate, so that the treatment is difficult.

The stem cell transplantation is considered to be one of the most promising methods for repairing nerve injury, but a lot of medicines and components for repairing nerve injury have different treatment effects, and an effective means is not available for how to select specific components to enable the components to be combined to exert a better technical effect and how to perform local efficient application on the components.

Disclosure of Invention

In order to solve the technical problems, the invention provides a stem cell gel for nerve repair and a preparation method thereof.

The preparation method specifically comprises the following steps:

(3) infecting the cells: adding the lentivirus carrying expressed G-CSF and SDF-1 proteins prepared in the step (2) into the P2 generation mesenchymal stem cells for infection to obtain mesenchymal cells expressing G-CSF and SDF-1;

Further, the G-CSF-2A-SDF-1 fragment was cloned into pRRLSIN. cPPT virus expression vector in step (1) at NheI and XbaI sites.

Further, the structure of the plasmid sequence in step (1) is as follows:

pLenti-CMV-G-CSF-2A-SDF-1-EGFP-3FLAG-PGK-Puro。

further, the mass ratio of the plasmid sequence to psPAX2 and PMD2.G in the system in step (2) is as follows: plasmid sequence psPAX2 pmd2.g ═ 4:2-5: 1-3.

Further, in the infected cells in the step (3), 48 hours after infection, the positive rate of SDF-1 was 58.2%, and the rate of SDF-1 positive cells was increased to 92.2% by puromycin screening for 12 days.

Further, in the gel material prepared in the step (4), the concentration of the sodium alginate aqueous solution is 0.1g/L, and the concentration of the calcium chloride aqueous solution is 0.1 g/L.

Further, in the preparation of the gel material in the step (4), every 10ml of sodium alginate solution is resuspended in 1X 10⁸And (4) cells.

Further, in the step (4), the calcium chloride aqueous solution and the sodium alginate aqueous solution in which the cells are resuspended are mixed in equal volumes.

The invention also provides a stem cell gel for nerve repair, which is prepared by the method.

The invention also provides the application of the stem cell gel for nerve repair in the medicine for treating nerve injury.

The culture method provided by the invention has the following technical effects: the stem cell gel for nerve repair provided by the invention has a good treatment effect on nerve injury.

Drawings

FIG. 1 shows the ratio of SDF-1 positive cells before and after puromycin screening for infection by adding lentivirus expressing G-CSF and SDF-1 protein into P2 mesenchymal stem cells;

FIG. 2 shows the ratio of G-CSF positive cells before and after puromycin screening by adding lentivirus expressing G-CSF and SDF-1 protein into P2 generation mesenchymal stem cells for infection;

FIG. 3 is a microscopic view of the stem cell gel prepared in example 1;

FIG. 4 is a statistical graph of SFI values measured at 2, 4, 8, and 12 weeks after surgery for rats in each group;

FIG. 5 is a statistical plot of BBB scores determined at 2, 4, 8, and 12 weeks post-surgery for rats in each group;

in fig. 4 and 5, the abscissa indicates time, and the ordinate indicates score.

Detailed Description

Example 1

The embodiment provides a preparation method of stem cell gel for nerve repair, which comprises the following steps:

(1) plasmid sequence synthesis: the G-CSF-2A-SDF-1 fragment was provided by Beijing Rui Boxing Ke Biotechnology Co., Ltd, and was synthesized by the following method: the G-CSF gene sequence (shown as SEQ ID NO: 1) and 2A (2A is a 2A element from a poina fusca virus (TaV), the gene connected with 2A can be translated and expressed normally after being transferred into cells, and can be used as an effective tool for constructing a polycistronic vector for gene transfer) to be connected in series to synthesize a G-CSF-2A fragment (shown as SEQ ID NO: 2), and then the G-CSF-2A-SDF-1 fragment (shown as SEQ ID NO: 4) is connected in series to an SDF-1 gene sequence (shown as SEQ ID NO: 3) to synthesize the G-CSF-2A-SDF-1 fragment. Cloning the G-CSF-2A-SDF-1 fragment to a pRRLSIN. cPPT virus expression vector, wherein the selected enzyme cutting sites are NheI and XbaI, and a plasmid sequence is obtained;

wherein the structure of the plasmid sequence is as follows:

pLenti-CMV-G-CSF-2A-SDF-1-EGFP-3FLAG-PGK-Puro；

cloning of the G-CSF-2A-SDF-1 fragment into pRRLSIN. cPPT virus expression vector as follows:

the ligase used was Solution I ligase (Solution I ligase was DNA ligase from TAKARA);

connecting system:

5ul of pRRLSIN. cPPT virus expression vector was used in combination with G-CSF-2A-SDF-1 fragment, and G-CSF-2A-SDF-1 fragment was 1:3-1: 10;

Solution I 5ul

temperature conditions (Pcr instrument):

65° 3min

4° 3min

16° 35min

the Solution I time is controlled to be 30min-60 min;

glue separation

③ running rubber

0.70g agarose, 90ml 1 TAE, 4ul GoodView, running voltage of 120V, marker and sample;

wherein, agarose is purchased from Saimerfin, and TAE Buffer solution (TAE Buffer) is composed of trihydroxymethyl aminomethane, acetic acid and ethylenediamine tetraacetic acid;

the preparation method of 50 × TAE Buffer comprises the following steps:

weigh Tris 242 grams, EDTA18.612 grams into a1 liter beaker;

adding about 800 ml of deionized water into a beaker, and fully and uniformly stirring;

adding 57.1 ml of glacial acetic acid, and fully dissolving;

adjusting pH to 8.3 with NaOH, adding deionized water to constant volume to 1 liter, storing at room temperature, and diluting 100 times to obtain 1 × TAE;

GoodView nucleic acid dye, available from baisheng gene technology ltd, beijing;

marker (DNA marker), DL2000 Plus, purchased from semer flyer;

cutting glue

Cutting the slowest strip, subpackaging with 1.5ml centrifuge tube, weighing, each between 0.2g and 0.3 g;

recovery of Gel (Gel Extraction Kit)

Adding QG (0.1g to 300ul) with three times of volume, carrying out vortex dissolution at 55 ℃ for 10min, and separating 3min to assist dissolution or peptizing;

adding isopropanol with the volume being one time of that of the sample, and slightly swirling or reversing the mixture to mix the mixture evenly;

thirdly, passing through the column, adding the solution into the column in several times, standing for 3-5 minutes, 12000rmp, 2 min; the recovery efficiency of general glue is low, and the glue can be recovered by using one column;

fourthly, washing, adding 750ul of PE, standing for 3-5 minutes, 12000rmp, and 2 minutes;

fifthly, repeating the previous step;

12000rmp, idling for 5 minutes, uncovering and airing for 5-10min to ensure that the ethanol is completely volatilized;

seventhly, dripping 30ul of EB to the center of the membrane, standing for 5min, 12000rmp, 3-5min, and collecting a solution to obtain a plasmid sequence;

wherein the gel recovery kit is purchased from Sigma-Aldrich company; QG, PE and EB are all names of reagents in the kit;

(2) constructing a virus: the plasmid sequence, psPAX2 and PMD2.G are packaged into lentivirus by adopting a three-plasmid packaging system, the total mass of DNA used for packaging the lentivirus is 30 mu g, HEK-293T cells are used as packaging cells of the lentivirus, the transfection method is calcium phosphate transfection, and the mass ratio of the plasmid sequence, psPAX2 and PMD2.G in the system is as follows: the plasmid sequence is psPAX2, PMD2.G is 4:3:2, namely the slow virus carrying protein expressing G-CSF and SDF-1 is obtained;

(3) infecting the cells: mu.l of the lentivirus prepared in step (2) was added to a total of 1.5X 10⁵The P2 generation mesenchymal stem cells were infected, and 48 hours after infection, selection was performed by puromycin; obtaining the mesenchymal cells expressing G-CSF and SDF-1; wherein, the ratio of SDF-1 positive cells before and after screening is shown in figure 1, the ratio of G-CSF positive cells before and after screening is shown in figure 2, the ratio of SDF-1 positive cells after lentivirus infection is 58.2%, the ratio of G-CSF positive cells is 48.4%, the ratio of SDF-1 positive cells after screening for 12 days by puromycin is increased to 92.2%, and the ratio of G-CSF positive cells is increased to 97.2%. (ii) a

(4) Preparing a gel material: dissolving 1g sodium alginate in 100ml pure water, stirring to dissolve, autoclaving to obtain sodium alginate water solution, mixing with 1 × 10⁸Resuspending the mesenchymal cells expressing G-CSF and SDF-1 in 10ml of sodium alginate aqueous solution to prepare sodium alginate aqueous solution with the cells resuspended, dissolving 1G of calcium chloride in 100ml of pure water, autoclaving to prepare calcium chloride aqueous solution, and uniformly mixing the calcium chloride aqueous solution and the sodium alginate aqueous solution with the cells resuspended to obtain stem cell gel;

the mesenchymal stem cells used in all the embodiments, the control examples and the test examples of the invention are umbilical cord mesenchymal stem cells, the umbilical cord mesenchymal stem cells are obtained by separating and culturing umbilical cords, the source of the umbilical cords is subsidiary hospitals of Changchun traditional Chinese medicine university, and the preparation method of the umbilical cord mesenchymal stem cells comprises the following steps:

preparing primary cells:

taking out the umbilical cord, and keeping the preservation solution in the collection bottle;

washing the umbilical cord: transferring umbilical cord to a 10cm sterile culture dish by using sterile toothed forceps, adding 75% alcohol to immerse the whole umbilical cord, sterilizing for no more than 2min, transferring to a new dish, adding 20ml of DPBS/PBS, washing, and repeating for 3 times to remove blood stain;

shearing umbilical cord into sections of about 3cm by using a sterile tissue shear, adding 20ml of DPBS/PBS to wash blood clots, and repeatedly washing for 3 times until blood stains are basically removed, wherein the washing liquid is clear;

removing blood vessels: transferring the umbilical cord segment into an empty sterile culture dish by using sterile toothed forceps, tearing a small opening at a position between an umbilical vein and an umbilical artery at one end of the umbilical cord segment, cutting the vein open, and removing the wall of the vein vessel to ensure that the Wharton's jelly layer is completely exposed on the surface;

separating the Walsh gum: the Wharton's jelly was torn off with a toothed forceps (taking care not to bring arterial vessels), placed in a sterile plate, 20ml of DPBS/PBS was added, and the jelly was washed.

Washing colloid: transferring the obtained colloid to another culture dish, adding 20ml of DPBS/PBS, washing off blood cells, and repeating for 1 time;

tissue homogenization: shearing the tissue into tissue homogenate blocks by using sterile tissue scissors;

dividing the tissue homogenate block into two parts, wherein one part is used for culturing primary cells, and the other part is used for freezing and storing tissues (the tissue block needing freezing and storing can be firstly centrifuged according to 4.3.12);

adding a proper amount of culture solution into the tissue homogenate blocks for culturing primary cells, uniformly blowing and beating, averagely inoculating into a T75 culture bottle, inoculating about 30 tissue homogenate blocks into each T75 culture bottle, and supplementing 5ml of culture solution for culture;

put the blake bottle flatly, rock a few times gently, make tissue piece evenly distributed as far as possible in whole bottom surface, place the blake bottle in carbon dioxide constant temperature and humidity incubator, culture condition: 37 plus or minus 0.5 ℃ and the volume fraction of carbon dioxide is 5 plus or minus 0.2 percent;

liquid supplementing:

observing the culture bottle after culturing umbilical cord Wharton's jelly till 3 days, supplementing liquid according to the condition, and if the liquid turns yellow or the bottom of the bottle becomes dry, supplementing 2ml of liquid (ensuring that the tissue block does not float up), and continuing culturing;

liquid changing:

performing primary full-scale liquid change until about 8 days, pouring out the old culture medium, supplementing 10ml of fresh culture medium, and placing in a carbon dioxide constant-temperature constant-humidity incubator for continuous culture;

primary collection:

on the 12 th day of umbilical cord Wharton's jelly culture, 2 culture bottles are taken to observe whether the cell morphology is abnormal or not and whether the cells are polluted or not under an inverted microscope, and digestion and harvest can be carried out if the cell fusion degree is observed to reach 70% -80%;

old medium was collected: slightly shaking the culture bottle to enable the tissue blocks with weak adherence to fall off, opening the cover, transferring the old culture medium and the fallen tissue blocks to a 50ml centrifuge tube, and combining 2-3 bottles into 1 tube;

slightly washing the cell culture bottle for 2 times by using DPBS/PBS;

digestion: 0.125% pancreatin-EDTA (ethylene diamine tetraacetic acid) is added into the washed culture bottles, 1ml of each bottle, and the culture bottles are overlaid by shaking lightly, so that the digestive juice of each culture bottle can infiltrate the bottom surfaces of the culture bottles. Standing in a super clean bench for 1min, taking out the culture bottle, patting slightly, observing under an inverted microscope, and when cells under the microscope become round, most of the cells fall off to indicate that digestion can be stopped;

termination of digestion: adding a digestion stop solution (primary cells are stopped by complete culture medium) into a culture bottle capable of stopping digestion, stopping digestion by 5ml each, transferring the suspension of the cells and the tissue blocks in the culture bottle after the digestion is stopped into a 50ml centrifuge tube, washing cell residues adhered in the culture bottle by 10ml of DPBS/PBS, and transferring the washed cell suspension into a 50ml centrifuge tube (adding pancreatin until the digestion is stopped, wherein the time is not more than 5 min);

centrifugal washing: placing the centrifugal tube into a centrifugal machine after balancing, and centrifuging for 5min at 1500 rpm;

merging the cell pellets after centrifugation: discarding the washing supernatant after centrifugation, suspending the cells by using a proper amount of complete culture medium, gently blowing, uniformly mixing, and finally merging the resuspended cell suspension into 150 ml centrifuge tube;

cell passage:

the number of generations: primary cells were passaged 1 or 2T 175 flasks by 1T 75 flask (depending on cell number);

arranging required T175 culture bottles into a row according to the number of the final passage T175 culture bottles and the capacity of the clean workbench for placing the T175 culture bottles at one time, vertically placing the required T175 culture bottles in the clean workbench, opening a cover after marking information such as cell codes, cell algebra, culture time and the like on the bottle wall, adding a culture medium into the bottle by using a 25ml pipette, wherein each culture medium is 23 ml;

a bottle is planted: after the cells are blown evenly, respectively adding 2ml of cell suspension into each T175 culture flask added with 23ml of culture medium, so that the final volume of the cells and the culture medium in each T175 culture flask is 25 ml; covering, horizontally placing and shaking up: after the cell suspension is added, the T175 culture bottle is covered and screwed, the culture bottles are horizontally placed in a stack of at most 4 cells, and the bottles are uniformly shaken for 10sec, so that the added cell suspension can be uniformly distributed on the bottom surface of the whole culture bottle. The first passage was designated as P1;

placing the mixture into a CO2 incubator for culture: stacking 3 evenly shaken T175 culture bottles in a CO2 incubator, and culturing at 37.0 + -0.5 deg.C, 5.0 + -0.2% CO2 concentration and saturated humidity until the cell fusion degree reaches 85-90%;

the prepared stem cell gel is placed under an electron microscope for observation, the result is shown in figure 3, the pore canal in the carrier can be observed from the lower figure to be far smaller than 1 mu m, the cells wrapped in the carrier can not climb out of the biological material, and the paracrine effect can be exerted in situ.

Example 2

The embodiment provides a preparation method of stem cell gel for nerve repair, which comprises the following steps:

(1) plasmid sequence synthesis: same as example 1;

(2) constructing a virus: the plasmid sequence, psPAX2 and PMD2.G are packaged into lentivirus by adopting a three-plasmid packaging system, the total mass of DNA used for packaging the lentivirus is 30 mu g, HEK-293T cells are used as packaging cells of the lentivirus, the transfection method is calcium phosphate transfection, and the mass ratio of the plasmid sequence, psPAX2 and PMD2.G in the system is as follows: the plasmid sequence is psPAX2, PMD2.G is 4:5:1, namely the slow virus carrying protein expressing G-CSF and SDF-1 is obtained;

(4) preparing a gel material: dissolving 1g sodium alginate in 100ml pure water, stirring to dissolve, autoclaving to obtain sodium alginate water solution, mixing with 1 × 10⁸And (2) resuspending the mesenchymal cells expressing G-CSF and SDF-1 in 10ml of sodium alginate aqueous solution to prepare a sodium alginate aqueous solution with the cells resuspended, dissolving 1G of calcium chloride in 100ml of pure water, performing autoclaving to prepare a calcium chloride aqueous solution, and uniformly mixing the calcium chloride aqueous solution and the sodium alginate aqueous solution with the cells resuspended to obtain the stem cell gel.

Example 3

The embodiment provides a preparation method of stem cell gel for nerve repair, which comprises the following steps:

(1) plasmid sequence synthesis: same as example 1;

(2) constructing a virus: the plasmid sequence, psPAX2 and PMD2.G are packaged into lentivirus by adopting a three-plasmid packaging system, the total mass of DNA used for packaging the lentivirus is 30 mu g, HEK-293T cells are used as packaging cells of the lentivirus, the transfection method is calcium phosphate transfection, and the mass ratio of the plasmid sequence, psPAX2 and PMD2.G in the system is as follows: the plasmid sequence is psPAX2, PMD2.G is 4:2:3, namely the slow virus carrying and expressing G-CSF and SDF-1 protein;

(4) preparing a gel material: dissolving 1g sodium alginate in 100ml pure water, stirring to dissolve, autoclaving to obtain sodium alginate water solution, mixing with 1 × 10⁸Resuspending the mesenchymal cells expressing G-CSF and SDF-1 in 10ml of sodium alginate aqueous solution to obtain sodium alginate aqueous solution with resuspended cells, dissolving 1G of calcium chloride in 100ml of pure water, autoclaving to obtain calcium chloride aqueous solution, and adding chlorine to the above solutionAnd uniformly mixing the calcium dissolving water solution and the sodium alginate water solution with the cells in a suspended manner to obtain the stem cell gel.

Comparative example 1

Comparative example 2

This comparative example provides a method for preparing a stem cell gel, which is different from example 1 in that the steps for synthesizing a plasmid sequence are as follows: obtaining SDF-1 gene sequence by gene synthesis method, cloning SDF-1 gene sequence to pRRLSIN. cPPT virus expression vector to obtain plasmid sequence, wherein SDF-1 gene sequence fragment is provided by Beijing Rui Boxing Ke Biotechnology GmbH.

Comparative example 3

This control example provides a method for preparing a stem cell gel, which is different from example 1 in that in control example 3, a nerve growth factor gene sequence (shown in SEQ ID NO: 5) obtained from Lemna minor of the Evodia department of the university of Wuhan's Hospital, Evodia, was cloned into pRRLSIN. cPPT virus expression vector in place of a fragment of G-CSF-2A-SDF-1.

Test example 1 therapeutic test on sciatic nerve defects in rats

Taking 100 SD rats with the age of 3 months, dividing the SD rats into a healthy control group and a model group, taking 10 rats except the healthy control group and taking other 90 rats as the model group, establishing a sciatic nerve injury model of the rats by using a clamp method for the rats of the model group, and operating the method as follows: performing intraperitoneal injection of 10% chloral hydrate for anesthesia, fixing, after conventional skin preparation and disinfection, carrying out blunt separation on left thigh muscle, exposing sciatic nerve, clamping the sciatic nerve at the root of the thigh by an aneurysm clip for 10s, repeating the clamping for 3 times to cause a 3mm injury area, observing continuity of an outer membrane and interruption of nerve axon by a microscope in the operation process, cleaning wounds by saline containing antibiotics, suturing tissues, and after the operation, obviously weakening the bending and stretching force by virtue of slow reaction of the left lower limb of a rat, serving as a standard for successful model creation, exposing the sciatic nerve of a healthy control group rat without clamping the injured nerve and then suturing the tissues; the unqualified model-made individuals were removed, 80 of the remaining qualified rats were selected and divided into a lesion group, example 1-3 groups, comparative example 1-3 groups and normal cell groups, each group consisting of 10 cells, according to the random number table method.

After operation, the stem cell gel of the group of examples 1-3, the stem cell gel of the group of comparative examples 1-3 and the mesenchymal stem cells without transgene modification are respectively implanted into the sciatic nerve injury of rats of the group of examples 1, the group of comparative examples 1-3 and the common cell group by using a micro-syringe, the healthy control group and the injury group are respectively injected with the same amount of physiological saline, the injection is carried out for 3min, a needle is left for 5min, and after the injection is finished, the rats are continuously fed for 12 weeks.

Sciatic nerve function index (SFI) assessment method was as follows:

self-make a 60cm long, wide 10cm, height 10 cm's both ends opening crib, lay in the tank bottom after cutting 70g white paper into and being as long as the equal width of crib, after the painting is soaked in the double ankle joint with pigment to the two hind limbs of rat, put the rat in one end of groove, make it walk to another party of groove by oneself, each side hind limb leaves 5-6 footprints respectively, select the clear footprint of mark to measure 3 indexes of normal foot (N) and wound side foot (E) respectively: A. PL (footprint length); B. TS (toe width); C. IT (width of middle toe), and substituting the above index into Bain formula to calculate sciatic nerve function index. Bain formula:

SFI 109.5(ETS-NTS)/NTS-38.3 (EPL-NPL)/NPL +13.3 (EIT-NIT)/NIT-8.8, sciatic nerve function index SFI 0 is normal, and-100 is complete injury. The footprints of rats at 2, 4, 8, and 12 weeks (after cell transplantation) were collected for calculation and the statistical results are shown in fig. 4.

The rat motor function was evaluated as follows:

the motor function of the rat is evaluated by adopting a BassoBeattie Bresnahan (BBB) motor function scoring method, the rat model is subjected to blind scoring 2, 4, 8 and 12 weeks after the rat model is made (after cell transplantation), the BBB score is between 0 (completely losing the innervation function) and 21 (normal movement), and the test result is shown in a graph 5.

As is clear from FIGS. 4 and 5, the SFI groups of the rats in the whole comparison showed a large difference in time. The three groups of stem cell gel groups have the best treatment effect, the recovery degree is closest to that of a healthy control group, the treatment effects of the control examples 1-3 groups and the common cell group are sequentially reduced, and an approximate result can be obtained according to evaluation of a BBB motor function grading method, so that the stem cell gel prepared by the method provided by the invention has considerable treatment effect on nerve injury.

Therefore, the invention is not limited to the specific embodiments and examples, but rather, all equivalent variations and modifications are within the scope of the invention as defined in the claims and the specification.

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