阅读说明：本技术 硫化氢缓释有机供体adt-oh在制备神经系统疾病治疗药物中的应用 (Application of hydrogen sulfide slow-release organic donor ADT-OH in preparation of medicine for treating nervous system diseases ) 是由 马艳霞 韦善文 邹明明 倪莉 周立宇 李迪 杨严伟 于 2021-09-07 设计创作，主要内容包括：本发明涉及硫化氢缓释有机供体ADT-OH药物在神经前体细胞分化中的应用,该物质能够诱导神经前体细胞更多的定向分化为神经元和少突胶质细胞,而更少的分化为星型胶质细胞,且促进神经元的轴突生长,同时可使神经前体细胞的死亡数减少,为移植神经前体细胞修复受损神经提供了方向,ADT-OH将有很大可能成为临床上治疗神经系统疾病的新药物靶点。(The invention relates to an application of a hydrogen sulfide slow-release organic donor ADT-OH medicament in nerve precursor cell differentiation, which can induce nerve precursor cells to be more directionally differentiated into neurons and oligodendrocytes and less differentiated into astrocytes, promote the axon growth of the neurons, reduce the death number of the nerve precursor cells, provide a direction for transplanted nerve precursor cells to repair damaged nerves, and can greatly become a new medicament target spot for clinically treating nervous system diseases.)

1. Application of hydrogen sulfide sustained-release organic donor ADT-OH in preparation of medicines for treating nervous system diseases.

2. Use according to claim 1, characterized in that: the hydrogen sulfide slow-release organic donor ADT-OH is used for repairing the nervous system.

3. Application of hydrogen sulfide slow-release organic donor ADT-OH in neural precursor cell in-vitro differentiation.

4. Use according to claim 3, characterized in that: the hydrogen sulfide slow-release organic donor ADT-OH is used for inducing neural precursor cells to differentiate into neurons.

5. Use according to claim 3, characterized in that: the hydrogen sulfide slow-release organic donor ADT-OH is used for promoting the growth of neuron axons.

6. Use according to claim 3, characterized in that: the hydrogen sulfide slow-release organic donor ADT-OH is used for inducing neural precursor cells to differentiate into oligodendrocytes.

7. Use according to claim 3, characterized in that: the hydrogen sulfide slow-release organic donor ADT-OH is used for inhibiting the neural precursor cells from differentiating into astrocytes.

8. Use according to any one of claims 3 to 7, characterized in that: the neural precursor cells are embryonic stage subconjunctival area neural precursor cells.

9. A method of inducing differentiation of neural precursor cells, comprising the steps of:

culturing neural precursor cells in neural precursor cell culture medium containing ADT-OH for 1-3d, and inducing culture in differentiation medium containing ADT-OH for 5-8 d;

the concentration of ADT-OH in the ADT-OH-containing neural precursor cell culture medium is 70-90 mu mol/L, the concentration of ADT-OH in the ADT-OH-containing differentiation culture medium is 70-90 mu mol/L, the neural precursor cell culture medium is DMEM-F12 culture medium containing B27, bEGF and FGF, and the differentiation culture medium is DMEM-F12 culture medium containing FBS and B27.

10. The method of claim 9, wherein: taking brain tissue under the ventricular duct membrane in the embryonic stage, digesting with pancreatin, terminating digestion with a neural precursor cell culture medium to obtain free neural precursor cells, taking the precipitate, adding the neural precursor cell culture medium, inoculating the precipitate into a cell culture plate, and continuously culturing for 3-4d to obtain the neural precursor cells.

Technical Field

The invention relates to the fields of biological cytology and regenerative medicine, in particular to application of a hydrogen sulfide slow-release organic donor ADT-OH in preparation of a medicine for treating nervous system diseases.

Background

Neural stem cells, also known as Neural Precursor Cells (NPCs), are cells with multipotential differentiation potential. During development of the Central Nervous System (CNS), the subventricular canalicular region is the region where NPCs are primarily distributed. NPCs can maintain self-cell numbers by symmetrical division and can also produce neurons, astrocytes and oligodendrocytes by asymmetrical division. In the mammalian nervous system, neurons are terminally differentiated cells that cannot re-divide themselves and give rise to new cells, and thus are essentially unable to replenish after neuronal death. The number of neural stem cells in the nervous system of adult mammals is low and is generally insufficient to compensate for neuronal death caused by nerve injury or disease. It is because of this neuronal deficit that damage or disease to the nervous system can lead to irreversible loss of sensory, motor, or cognitive function. Therefore, how to generate new neurons and re-form functional circuits is an important goal in current neuroscience research and future clinical treatments for nerve injury repair and regeneration.

After nerve injury, some glial cells, such as astrocytes, are reactivated and rapidly divide after injury, and are replenished quantitatively. However, these newly formed astrocytes are often the main components of scar tissue and act as inhibitors of axonal regeneration after nerve injury. Oligodendrocytes are axon myelin sheath cells that are generally replenished by division and differentiation of precursor cells after injury. However, the damaged nerve tissue has a certain inhibiting effect on the formation of new myelin sheaths, so that the formation of new myelin sheaths cannot be completely satisfied by endogenous oligodendrocytes. Therefore, repairing the damaged nervous system and restoring function requires: 1) Replenishment of new neurons, 2) replenishment of new oligodendrocytes, and 3) reduction of astrocytes forming scar tissue.

Transplantation of neural precursor cells to repair the damaged nervous system is an important research direction in the field of neural repair. However, current research in the art indicates that the neural precursor cells transplanted to the damaged site are largely differentiated into astrocytes to form glial scars, and thus the effect of repairing the damaged nerves is not achieved. Therefore, how to induce transplanted exogenous stem cells into neurons or oligodendrocytes is an important research direction in the field of neuroscience at present.

Hydrogen sulfide (H)₂S) is a third known endogenous gas signal molecule following nitric oxide and carbon monoxide, expressed in various organs of mammals, especially the brain. H₂S has protective effect on ischemia reperfusion injury of liver and other organs. In addition, it is involved in various physiological and pathological processes, and regulates the polarization of central microglia from m 1-type activation state to m2 activation state, thereby reducing inflammatory response of central nervous system and enhancing the regeneration and repair of injured brain. Due to H₂S has the characteristic of protecting cells and can artificially regulate endogenous H₂S or biosynthesis of H₂S donors are administered in vitro to restore physiological function to the cell or organ system. Therefore, interest is increasingly focused on identifying new H₂And (3) an S donor. Due to H₂The concentration of S is rapidly increasing in time to be lethal, so that a donor with a slow release rate is required. 5- (p-hydroxyphenyl) -1, 2-dithione-3-thione (ADT-OH) is a substance capable of releasing H slowly in vivo₂An organic donor of S. With inorganic H such as NaHS₂Compared with S donor, ADT-OH can slowly and permanently release H at a controllable rate₂S and the effect is superior to that of common inorganic H₂And (3) an S donor. ADT-OH releases H in vivo₂The S molecule has a neuroprotective effect on oxidative stress induced by glutamic acid. However, the potential effect of ADT-OH on neural precursor cell differentiation is not clear.

Disclosure of Invention

In order to solve the technical problems, the invention provides a new application of a hydrogen sulfide slow-release organic donor ADT-OH, the substance can reduce the death number of neural precursor cells, can induce more neural precursor cells to directionally differentiate into neurons and oligodendrocytes and less neural precursor cells to differentiate into astrocytes, provides a direction for transplanting the neural precursor cells to repair damaged nerves, and the ADT-OH can be a new drug target spot for clinically treating nervous system diseases.

The invention claims application of a hydrogen sulfide slow-release organic donor ADT-OH in preparation of a medicine for treating nervous system diseases.

Further, the hydrogen sulfide slow-release organic donor ADT-OH is used for repairing a nervous system, wherein the ADT-OH induces neural precursor cells to be differentiated into neurons and oligodendrocytes and inhibits the neural precursor cells from being differentiated into astrocytes.

The invention also claims application of the hydrogen sulfide slow-release organic donor ADT-OH in vitro differentiation of neural precursor cells.

Further, the hydrogen sulfide slow-release organic donor ADT-OH is used for inducing the neural precursor cells to be differentiated into neurons.

Further, the hydrogen sulfide slow release organic donor ADT-OH is used to promote neuronal axonal growth.

Further, the hydrogen sulfide slow-release organic donor ADT-OH is used for inducing neural precursor cells to differentiate into oligodendrocytes.

Further, the hydrogen sulfide slow-release organic donor ADT-OH is used for inhibiting the neural precursor cells from differentiating into astrocytes.

Furthermore, the nerve precursor cells are embryonic cerebral cortical nerve precursor cells, in particular ventricular duct subepithelial nerve precursor cells. The present invention uses embryonic-stage subventricular ductal nerve precursor cells because the number of neural stem cells in the adult mammalian nervous system is small and generally insufficient to compensate for neuronal death caused by nerve injury or disease. A small number of neural precursor cells in the subcapillary area of the adult mammalian brain ventricles can also differentiate into neurons and glial cells, but it is difficult to achieve the effect of repairing nerve damage due to insufficient numbers thereof.

The neural precursor cell under the ventricular duct membrane is also called neural stem cell, and is a cell capable of self-renewal and having multidirectional differentiation potentialThe neural progenitor cells can proliferate and differentiate into neurons and glial cells, and before the 12 th day of embryonic development, the neural progenitor cells proliferate in large quantities to form a cell pool, which provides conditions for the development of the later cerebral cortex. To eliminate ethical issues, when applied to humans, the present invention uses Human Neural Progenitor Cells obtained with ethical approval, including commercial Human Neural Progenitor Cells (e.g., NHNP-Human Neural progenerator Cells, HopCell)^TMEtc.). In particular, none of the neural precursor cells used in the present invention can develop into an intact individual.

A method of inducing differentiation of neural precursor cells, comprising the steps of:

culturing neural precursor cells in neural precursor cell culture medium containing ADT-OH for 1-3d, and inducing culture in differentiation medium containing ADT-OH for 5-8 d;

the concentration of ADT-OH in the ADT-OH-containing neural precursor cell culture medium is 70-90 mu mol/L, the concentration of ADT-OH in the ADT-OH-containing differentiation medium is 70-90 mu mol/L, the neural precursor cell culture medium is DMEM-F12 medium containing B27, bEGF and FGF, and the differentiation medium is DMEM-F12 medium containing FBS and B27.

Further, taking brain tissue under ventricular duct membrane in embryo stage, digesting with pancreatin, terminating digestion with neural precursor cell culture medium to obtain free neural precursor cell, taking precipitate, adding neural precursor cell culture medium, inoculating into cell culture plate, and continuously culturing for 3-4d to obtain the neural precursor cell;

preferably, the concentration of ADT-OH is 80. mu. mol/L.

By the scheme, the invention at least has the following advantages:

in order to solve the problem of how to induce the directional differentiation and survival of the neural precursor cells, the invention innovatively treats the neural precursor cells cultured in vitro through ADT-OH medicaments, provides a medicament and a method for inducing the directional differentiation and protecting the survival of the neural precursor cells, achieves the purposes that the neural precursor cells are more differentiated into neurons and oligodendrocytes required by the repair of neural injury, less differentiated into astrocytes and the death number of the neural precursor cells is reduced, and lays a foundation for adopting the transplantation of the neural precursor cells to repair the damaged nerves.

The foregoing description is only an overview of the technical solutions of the present invention, and in order to make the technical solutions of the present invention more clearly understood and to be implemented according to the content of the description, the following description is made with reference to the preferred embodiments of the present invention and the detailed drawings.

Drawings

In order that the present disclosure may be more readily and clearly understood, reference will now be made in detail to the present disclosure, examples of which are illustrated in the accompanying drawings.

FIG. 1 shows the effect of ADT-OH on neuron differentiation, wherein A is Tuj1 cell staining pattern, B is neuron differentiation ratio statistical pattern, and C is the neuron axon length quantitative result;

FIG. 2 shows the effect of ADT-OH on oligodendrocyte differentiation, wherein A is MBP cell staining pattern and B is statistical plot of oligodendrocyte differentiation ratio;

FIG. 3 is a graph showing the effect of ADT-OH on astrocyte differentiation, wherein the left graph is a GFAP cell staining graph, and the right graph is a statistical graph of the differentiation ratio of astrocytes;

FIG. 4 shows the effect of ADT-OH on astrocyte differentiation, wherein A is caspase3 cell staining pattern and B is a statistic map of neural precursor apoptosis.

Detailed Description

The present invention is further described below in conjunction with the following figures and specific examples so that those skilled in the art may better understand the present invention and practice it, but the examples are not intended to limit the present invention.

Example 1

(1) Adult ICR mice were mated randomly in a 2:1 ratio. Male and female mice were housed at 8 pm daily. Day 2, mating success was indicated if female mice had a vaginal plug at 8 am, which day was recorded as the first day the mice were pregnant. When the mouse is pregnant for 14.5 days (E14.5), the mouse can be used for neural precursor cell extraction.

C57BL/6J mouse pregnant for 14.5 days is anesthetized by intraperitoneal injection with 4% chloral hydrate, the abdominal cavity of the pregnant mouse is opened by sterile surgical scissors and surgical forceps, the uterus of the pregnant mouse is planed to take out the brain of the fetal mouse, isolating fetal rat brain tissue under a microscope, digesting the tissue with 0.05% pancreatin in a water bath at 37 deg.C for 10min, terminating digestion with neural precursor cell culture medium (DMEM-F12 medium 39.2ml, 1X B27800 ul,0.2mg/ml bEGF 4ul,0.2mg/ml FGF 8ul), then gently blowing the tissue into single cells, centrifuging the cells at 700rcf for 5 minutes, removing supernatant, adding a neural precursor cell culture medium, inoculating the neural precursor cell culture medium into a 24-hole cell culture plate for culture, and carrying out next detection when the cells grow for 3-4 days and proliferate to sufficient numbers.

When the cultured primary nerve precursor cells grow to a certain size of nerve cells, the nerve cells are blown into single cells and then inoculated into a 24-well cell culture plate paved with an adhesive disk slide, after the cells grow for 24 hours in the nerve precursor cell culture medium added with ADT-OH (80 mu mol/L) and DMSO as control solutes, the nerve precursor cell culture medium is respectively changed into a differentiation culture medium added with ADT-OH and DMSO (DMEM/F123 1239 ml, 0.5% FBS,1X B27800 ul) to induce the differentiation of the nerve precursor cells. Then the next detection is carried out.

Cultured cells were fixed with 4% PFA for 15 minutes, washed 3 times with 0.01M PBS and immunofluorescent-stained with Tuj1, GFAP, MBP, caspase3 antibodies. Mounting was performed with a mounting medium containing DAPI (Vector Labs, H-1400). Observed under a Zeiss fluorescent microscope and photographed. ImageJ and adobe photoshop were used to count Tuj 1-positive neurons, GFAP-positive astrocytes, MBP-positive oligodendrocytes, and DAPI-positive cells, respectively. The proportion of positive cells to all DAPI positive cells was calculated.

(2) After the cells are differentiated in a differentiation medium for 5 days, 4% PFA is used for fixing the cells, cell immunofluorescence staining is carried out by using a specific antibody Tuj1 of the neurons, after the staining is finished, picture collection is carried out under a fluorescence microscope, and the ratio of each group of neurons in all DAPI positive cells is counted.

The results are shown in FIG. 1. Statistics show that more cells in the ADT-OH group differentiated into neurons (FIGS. 1A and B) and the axons of the neurons were longer (FIGS. 1A and C) compared to the DMSO control group. The above data show that ADT-OH can promote neuronal differentiation and axonal growth of neural precursor cells.

(3) After the cells were differentiated in the differentiation medium of neural precursor cells for 5 days, the cells were fixed with 4% paraformaldehyde, immunofluorescent staining was performed with an antibody specific to oligodendrocytes, and the ratio of MBP-positive cells to all DAPI-positive cells was counted.

The results are shown in FIG. 2. Statistics show that more cells differentiated into oligodendrocytes in the ADT-OH group compared to the DMSO control group (FIGS. 2A and B), and the data show that ADT-OH promotes differentiation of neural precursor cells into oligodendrocytes.

(4) After the cells are differentiated in a differentiation medium of neural precursor cells for 8 days, the cells are fixed by 4% paraformaldehyde, immunofluorescence staining is carried out by using a specific antibody GFAP of astrocytes, and the ratio of GFAP positive cells to all DAPI positive cells is counted.

The results are shown in FIG. 3. Statistics show that the ADT-OH group has less cells to differentiate into astrocytes compared with the DMSO control group, and the data show that ADT-OH can inhibit differentiation of neural precursor cells into astrocytes.

Example 2

After 3-4 days of culture of primary isolated neural precursor cells, the neurospheres are blown into single cells, the single cells are inoculated into 24-hole cell culture plates paved with adhesive slides, the cells are respectively stained with an apoptosis specific antibody caspase3 after 3 days of growth in neural precursor cell culture media added with ADT-OH (80 mu mol/L) and a control solute DMSO, the ratio of caspase3 positive cells to all DAPI positive cells is counted after pictures are collected under a fluorescence microscope, the statistical result shows that the ADT-OH group has fewer cells in apoptosis compared with a DMSO control group (figures 4A and B), and the data show that ADT-OH can protect the neural precursor cells from apoptosis.

In conclusion, compared with the DMSO control group, the neuron differentiation rate of the ADT-OH group is increased by 57.69%, the oligodendrocyte differentiation rate is increased by 87.5%, and the astrocyte differentiation rate is reduced by 69.2%. In addition, ADT-OH can also effectively prevent apoptosis of neural precursor cells, and compared with DMSO control group, apoptosis of ADT-OH group is reduced by 44.8%.

Example 3

Adopting the heavy object to fall and impact the dorsal side of the spinal cord of the mouse, establishing a spinal cord injury model of the mouse, and specifically operating as follows: anaesthetizing the mouse, cutting the center of the back of the mouse, cutting off the vertebral plate, and exposing the dura mater of the spinal cord; then the weight is freely dropped from a certain height and is hit on the spinal cord, so that the spinal cord is damaged. Then, the ADT-OH drug-treated neural precursor cells are transplanted to the spinal cord injury site under a body type microscope, and the differentiation, cell survival and neurite growth of the neural precursor cells at the injury site are detected after one month to evaluate the effect on spinal cord repair.

The result shows that after the neural precursor cells treated by the ADT-OH medicament are implanted for one month, the quantity of neurons and oligodendrocytes at the spinal cord injury part of a mouse is greatly increased, the quantity of astrocytes is obviously lower than that of the neurons or the oligodendrocytes, axons also extend to a certain extent, and the spinal cord functional injury is obviously improved.

It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications of the invention may be made without departing from the spirit or scope of the invention.