CN115975929A - Application of KAT2A inhibitor in treating spinal cord and sciatic nerve injury - Google Patents

Abstract

The invention belongs to the field of biology/tissue engineering, and relates to an application of a KAT2A inhibitor in treatment of spinal cord and sciatic nerve injuries, wherein the KAT2A inhibitor is MB-3, and the MB-3 is used for in vitro culture of neural stem cells, so that the proliferation and balling rate of the neural stem cells are obviously increased, and further, the spinal cord and sciatic nerve injuries are treated. The expanded neural stem cells after MB-3 treatment can still exert functions to promote the recovery of spinal cord and sciatic nerve injuries, can provide stable and controllable cell sources for clinical application of the neural stem cells, and has wide application prospects and market values.

Description

Application of KAT2A inhibitor in treating spinal cord and sciatic nerve injury

Technical Field

The invention belongs to the field of biology/tissue engineering, and mainly relates to a method for preparing tissue engineering nerve seed cells for treating spinal cord and sciatic nerve injury by adopting MB-3 and application thereof.

Background

The central nervous system and the peripheral nervous system form the complete nervous system of the human body, and the nervous system plays a leading role in maintaining the steady state of the internal environment of the organism, keeping the integrity and the uniformity of the organism and the coordination and the balance of the external environment. With the deepening of the modernization process of the society, traffic accidents, motion accidents and the like are increased inevitably, and the damage of the nervous system can be caused by frequent violent incidents and natural disasters. Currently, autologous nerve graft vegetation is considered as a gold standard for treating nerve defects, but the limited source of donor nerves and the defect of the nerve function of a donor area greatly limit the clinical application of the autologous nerve graft vegetation. In this case, tissue engineered nerves have been produced as an effective method for treating nerve damage.

The tissue engineering nerve graft is mainly composed of a biological material bracket, seed cells and neurotrophic factors. Stem cells can differentiate into many types of cells, including neurons and glial cells. In addition, may be harvested from autografts to reduce immunogenicity. Therefore, the stem cells have good clinical application prospect as seed cells for constructing tissue engineering nerves. The research finds that the nerve stem cells can effectively promote the facial nerve repair by combining the nerve stem cells with the nerve conduit compounded by the neurotrophic factor 3 and the hyaluronic acid-collagen, and another research finds that the nerve stem cells in the nerve conduit can stimulate Schwann cell differentiation and promote the functional recovery of damaged sciatic nerves by increasing the expression of IL12p 80. Although stem cells have encouraging repair effects as seed cells, potential tumorigenic risks exist, the stem cell source is limited, in vitro proliferation culture is difficult, the in vitro amplification quantity has obvious limitations, the use of stem cells/tissue engineering nerves is greatly limited, and therefore, an expandable stem cell source capable of providing enough quantity according to requirements is needed.

The KAT2A gene is also named GCN5 or GCN5L2, belongs to histone acetyltransferase family, mainly plays the gene transcription regulation function, and the transcription activity of the gene is highly conserved in the whole eukaryote. Acetylation of KAT2A is associated with chromatin regulation, autophagy, neuronal apoptosis, cell proliferation, chromosome condensation, inflammation, cell differentiation of stem cells, hematopoiesis, and oxidative stress. Have been extensively studied for their role in epigenetic regulation and pathogenesis of different types of diseases, such as cancer, diabetes, neurological diseases, infectious diseases, and aging. The KAT2A gene is involved in the production of peroxides and is thought to play an important regulatory role in immune responses, and acetylation modifying functions of the KAT2A gene are necessary to maintain normal cell cycle progression. The inventor firstly finds out in previous researches that KAT2A plays an important role in the process of maintaining the dryness of the neural stem cells, and the KAT2A activity is inhibited to promote the proliferation of the neural stem cells along with the gradual up-regulation of the expression of the KAT2A in the differentiation of the neural stem cells. MB-3 as KAT2A inhibitor is relatively lack of reports about the aspects of stem cell proliferation, dryness maintenance and application of MB-3 at present, and no report is made that MB-3 is used for preparing tissue engineering nerve seed cells to treat spinal cord and sciatic nerve injuries.

Disclosure of Invention

The invention aims to provide a relatively simple and effective stem cell proliferation culture method for preparing tissue engineering nerves for treating spinal cord and sciatic nerve injuries based on the current situation and research foundation of the prior art, and particularly relates to a method and application of MB-3 for preparing tissue engineering nerve seed cells.

The technical scheme adopted by the invention is as follows:

the KAT2A inhibitor is MB-3, and the MB-3 is used for in vitro culture of the neural stem cells, so that the proliferation and balling rate of the neural stem cells are obviously increased, and the spinal cord and sciatic nerve injuries are further treated.

Further, the method for culturing the neural stem cells in vitro comprises the following steps: using SD pregnant mouse, taking out fetal mouse, separating cerebral cortex, digesting with pancreatin to obtain single cell suspension, and culturing; MB-3 is continuously added during the culture process, and the culture and the passage are continuously carried out until a sufficient number of neural stem cells are obtained.

Further, the concentration of MB-3 was 100. Mu.M.

Further, the SD pregnant mice were pregnant for 14 days.

Further, MB-3 was continuously added during the culture, and the culture was continued until the stem cell balls had a diameter of about 150 μm, after which the cells were digested and passaged, and the passage was continued until a sufficient number of neural stem cells were obtained.

Further, the primer sequences for detecting changes in KAT2A RNA levels were:

GCN5-sense 5'TCATCGGTGGGATTTGCT 3'；

GCN5-antisens 5'TACTCGTCGGCGTAGGTG 3'。

furthermore, the neural stem cells cultured in vitro are mixed with the tissue engineering hydrogel for repairing spinal cord injury.

Further, the tissue engineering hydrogel is methacrylated silk fibroin.

Further, the concentration of the methacrylated silk fibroin is 5-20%.

Furthermore, the neural stem cells are cultured on the tissue engineering nerve conduit and then transplanted for injury repair of sciatic nerve.

Further, the step of constructing the nerve conduit is as follows: filtering 10% methylacryloyl acylated gelatin solution for sterilization, pouring into a catheter mould to make the inner diameter and the outer diameter of the catheter respectively 2mm and 4mm, freezing at-80 ℃ for 24h after ultraviolet irradiation curing, defrosting and demoulding after freeze-drying, and then co-culturing the neural stem cells and the catheter.

Further, the application comprises the following steps:

1. isolated and cultured neural stem cells

SD pregnant mouse (14 days of pregnancy, E14 d) is used, fetal mouse is taken out, cerebral cortex is separated, and single cell suspension is prepared for culture after trypsinization.

MB-3 treatment of neural Stem cells for expansion

Adding KAT2A inhibitor MB-3 continuously during the culture process, digesting and subculturing until the diameter of the stem cell ball is about 150 μm, and continuously culturing and subculturing until a sufficient number of neural stem cells are obtained.

3. Preparation of tissue engineered nerve grafts for treatment

After being mixed with tissue engineering hydrogel (such as methacrylated silk fibroin), the neural stem cells cultured in vitro can be used for repairing brain or spinal cord injury; the neural stem cells are cultured on the tissue engineering nerve conduit and then transplanted to be used for injury repair of sciatic nerves.

Advantageous effects

The invention provides a relatively simple and effective method for culturing and amplifying neural stem cells in vitro, when KAT2A inhibitor MB-3 is used, the proliferation and balling rate of the neural stem cells are obviously increased, the multidirectional differentiation potential is not affected, the continuous amplification of the neural stem cells in vitro can be simply and conveniently realized, and enough stem cells can be obtained only by a small amount of donor tissues. Meanwhile, dryness reduction caused by continuous expansion of the neural stem cells is also inhibited by MB-3. In addition, the neural stem cells treated by MB-3 can be more differentiated to neurons under the non-directional condition, which is beneficial to the recovery of the neural function. Finally, the expanded neural stem cells after MB-3 treatment can still exert the function to promote the recovery of spinal cord and sciatic nerve injuries, can provide stable and controllable cell sources for the clinical application of the neural stem cells, and has wide application prospect and market value.

Drawings

FIG. 1 shows the detection results of neural stem cell markers, (A) fluorescence staining of neural stem cells Nestin, (B) fluorescence staining of neural stem cells SOX2, (C) fluorescence staining of neural stem cells PAX6, and (D) statistical chart of the positive proportion of each marker;

FIG. 2 is a graph showing that KAT2A expression is gradually up-regulated during neural stem cell differentiation, (A) immunofluorescence staining patterns of neural stem cell differentiation 0, 24 and 48h KAT2A (GCN 5), (B) changes in neural stem cell differentiation 0, 24 and 48h KAT2A mRNA levels, (C) changes in neural stem cell differentiation 0, 24 and 48h KAT2A protein levels, and (D) statistical graphs of KAT2A protein level changes;

FIG. 3 is a photograph showing that MB-3 promotes the proliferation of neural stem cells, (A) is a DMSO control group, (B-D) is a MB-3 treatment group at various concentrations, and (E) is a statistical chart of EdU staining;

FIG. 4 shows that MB-3 increases the spheronization rate of neural stem cells;

FIG. 5 shows that the neural stem cells are more differentiated towards neurons after MB-3 treatment;

FIG. 6 is a photograph showing that MB-3 inhibits the differentiation process of neural stem cells, wherein (A) is a bright field map of a control group and an MB-3 treatment group which differentiate for 24 hours, 48 hours and 72 hours, and (B) is a statistical map of the axon lengths of neurons after differentiation;

FIG. 7 shows that the stem cells effectively promote the recovery of spinal cord injury after MB-3 treatment;

FIG. 8 shows that the stem cells after MB-3 treatment can effectively promote the recovery of sciatic nerve injury.

Detailed Description

Example 1

KAT2A expression as biomarker for neural stem cell dryness

1. Primary culture of neural stem cells

After the SD pregnant mouse (E14 d) was euthanized, a fetal mouse was taken out, a brain part was identified under a stereomicroscope and a membrane was peeled off, tissues such as hippocampus were removed, and a cerebral cortex was placed in 3mL of a previously prepared complete Medium (50 mL system, DMEM/F12: neuroBasal Medium (1:1) with 23.75mL of each of B27 (50X): 2%, N2 (100X): 2%, EGF: 1. Mu.g; bFGF: 1. Mu.g; P-S:1%, glutamax (100X): 1%) and lightly blown 30 mesh with a 200-mesh screen. And (5) blowing 60 times, diluting to complete culture, culturing at 37 ℃, and digesting and passaging after cells are pelleted.

2. Immunofluorescence neural stem cell marker expression

And (3) taking P2 to substitute the balled neural stem cells to be digested into single cells, putting 5-8 thousands of the cells on a glass slide according to each hole, and collecting samples according to time points. Cells were fixed with 4% paraformaldehyde, and washed 3 times with PBS after 30min, 5min each time. Blocking with cell blocking solution (GCB) at 37 ℃ for 1h. Diluting primary antibody (Nestin, sox2 and Pax 6) with antibody diluent to the final use concentration, incubating overnight at 4 ℃, rewarming for 20-30min at room temperature, washing for 3 times with PBS, preparing the required secondary antibody according to the primary antibody source, and incubating for 2 hours at room temperature in a dark place. Incubating Hoechst 33315min at room temperature, washing by PBS, sealing, photographing and measuring and counting, wherein the result is shown in figure 1, and the neural stem cells are successfully obtained.

3.Fibronectin induces neural stem cell differentiation, detects the expression change of KAT2A

pll stock with ddH ₂ O =1, 10 dilution coated petri dish, overnight at room temperature, ddH ₂ O washing for more than 5 times, and then coating with Fibronectin at the concentration of 30 μ g/mL for 2h at 37 ℃. Removing the Fibronectin, adding the treated cell suspension, and collecting samples at required time points after the adherent induction differentiation. qRT-PCR detects the change of KAT2A RNA level, westernBlot and immunofluorescence staining detects the change of protein level, as shown in figure 2, with the differentiation degree of the neural stem cell deepening, KAT2A protein and RNA level is gradually up-regulated, which shows that KAT2A expression level can reflect the dryness of the neural stem cell, and can be used as a drug target for maintaining the dryness of the stem cell.

Real-time primer sequences:

GCN5-sense 5'TCATCGGTGGGATTTGCT 3'

GCN5-antisens 5'TACTCGTCGGCGTAGGTG 3'

example 2

KAT2A inhibitor MB-3 for promoting neural stem cell proliferation

1. Culture of neural stem cells

The SD pregnant mouse (E14 d) is anesthetized and killed, the fetal mouse is taken out, the brain part is aligned under a stereoscopic microscope and is stripped, tissues such as hippocampus and the like are removed, the cerebral cortex is placed into 3mL of complete culture medium prepared in advance, the cells are lightly blown for 30 times, and the cells are screened by a 200-mesh screen. Blow 60 ℃ lower, dilute to complete culture medium and culture at 37 ℃.

2. Inhibition of KAT2A activity by MB-3

The cells were divided into two groups, a control group and a KAT2A inhibitor group, the stem cells of the control group were treated with DMSO in an amount consistent with that of MB-3, the stem cells of the KAT2A inhibitor group were treated with Butyrolactone 3 (MB-3), and MB-3 was a specific small molecule inhibitor of histone acetyltransferase KAT2A and was highly compatible with KAT 2A. The MB-3 concentration is 100 MuM, continuous passage is carried out to observe the change of the balling rate of each group of neural stem cells, the MB-3 is removed after each passage of cell generation for directional and non-directional induced differentiation, and the difference between the differentiation potential and the contrast group is observed. After MB-3 treatment, fibronectin induced differentiation was performed, and the influence of MB-3 on the proliferation and differentiation degree of neural stem cells was examined.

Effect of MB-3 on neural Stem cell proliferation

MB-3 treatment was followed by fibrinectin-induced differentiation, and EdU was used to detect the proliferation of stem cells, as shown in FIG. 3, MB-3 was able to promote the proliferation of neural stem cells.

3. Detecting the Effect of MB-3 on neural Stem cells

The MB-3 treated neural stem cells are subjected to continuous passage, the change of the balling rate of each generation of stem cells is observed, the neural stem cell masses of more than 50 μm are included in statistics, the balling rate = the number of the neural stem cell masses/the number of the inoculated cells, and the statistics are compared with a control group, and the result is shown in figure 4, wherein the balling rate of the MB-3 treated stem cells is higher than that of the control group, and the difference is gradually increased along with the increase of the generation number.

Effect of MB-3 on neural Stem cell differentiation

After removing MB-3, non-directional differentiation and Fibronectin directional differentiation are carried out, immunofluorescence staining is carried out, tuj1 marks neurons, GFAP marks astrocytes, photographing statistics is carried out, the influence of MB-3 on the differentiation potential of the neural stem cells is observed, as a result, the MB-3 has no influence on the differentiation potential of the neural stem cells, and statistics shows that the MB-3 treated neural stem cells are more differentiated towards the neurons when being not directionally differentiated (figure 5). The neuron protrusion length is photographed and counted after 24, 48 and 72 hours of differentiation, and the differentiation process of the neural stem cells is found to be inhibited compared with the control group.

Example 3

Preparation of tissue engineering nerve graft by using MB-3 for treating spinal cord injury

1. Seed cell preparation

The SD pregnant mouse (E14 d) is anesthetized and killed, the fetal mouse is taken out, the brain part is aligned under a stereoscopic microscope and is stripped, tissues such as hippocampus and the like are removed, the cerebral cortex is placed into 3mL of complete culture medium prepared in advance, the cells are lightly blown for 30 times, and the cells are screened by a 200-mesh screen. And (4) blowing and beating for 60 minutes, diluting to complete culture, culturing at 37 ℃, adding 100 mu M MB-3 in the culture process to treat and keep the stem cells dry, digesting and subculturing to amplify the neural stem cells after the cells form spheres until enough neural stem cells are obtained.

2. Establishment of spinal cord injury model

Anaesthetizing a female adult rat with the weight of 220g, preparing skin at the chest and back, carrying out blunt dissection on a right vertebral plate of T9 along an incision on a T10 spinous process, fixing peripheral tissue muscles, biting the vertebral plate of the spinous process and a right articular process, inserting a blade into a posterior median groove, transversely cutting to a central tube, removing a 3mm right spinal cord tissue, and at the moment, generating spasm and convulsion on the lower limb of one side of the rat and then paralysis.

Treating spinal cord injury by combining MB-3 treated seed cells with tissue engineering hydrogel

Experimental animals were divided into two groups, a control group using hydrogel only and an experimental group using seed cell-bound tissue engineering hydrogel, the experimental group mixing the neural stem cells obtained using MB-3 with 5% -20% (w/v) methacrylated silk fibroin solution (diluted with PBS containing photoinitiator LAP (0.25% w/v)) (EFL, suzhou), injecting the mixture to the lesion, curing the mixture by ultraviolet irradiation at 405nm wavelength for 10-30s, and suturing the wound. The control group was treated with methacrylated silk fibroin hydrogel only. The post-operative observation continued for 4 weeks, weekly animal behavioral tests (motor function tests, including TSE fine motor, BBB score, etc.). After 1 day, 3 days, 7 days and 14 days of operation, rat spinal cord tissues are fixed, and are subjected to conventional histochemical staining after being sliced, the growth and differentiation conditions of stem cells are observed, the results show that the neural stem cells treated by MB-3 can effectively promote the repair of spinal cord injury, the behavioural results are better than those of a control group as shown in figure 7, and the neural stem cells treated by MB-3 can still play the function of repairing the neural injury.

Example 4

Method for preparing tissue engineering nerve conduit for treating sciatic nerve injury by using MB-3

1. Seed cell preparation

The SD pregnant mouse (E14 d) is anesthetized and killed, then the fetal mouse is taken out, the brain part is aligned under a stereomicroscope and is stripped, tissues such as hippocampus are removed, the cerebral cortex is placed in a prepared 3mL complete culture medium, and the fetal mouse is lightly blown for 30 times and passes through a 200-mesh screen. And (3) blowing and beating for 60 minutes, diluting until complete culture is realized, culturing at 37 ℃, adding 100 mu M MB-3 in the culture process to maintain the dryness of the stem cells, and digesting and subculturing to amplify the neural stem cells after the cells form spheres until enough neural stem cells are obtained.

2. Establishment of sciatic nerve transection model

Female adult rats weighing 220g were anesthetized, the leg was preserved, tissue muscles were separated blunt, and sciatic nerves were exposed and cut off to construct sciatic nerve transection models.

3. Construction of tissue engineering nerve graft containing cells

First, a nerve conduit was constructed, 5-30% (w/v) (diluted with PBS containing photoinitiator LAP (0.25% w/v)) of a methacrylated gelatin solution was filtered and sterilized, and then poured into a conduit mold so that the inner and outer diameters of the conduit were 2mm and 4mm, respectively, after curing by ultraviolet irradiation for 10-30s, frozen at-80 ℃ for 24 hours, freeze-dried, then, demolded by defrosting, and then, the neural stem cells were co-cultured with the conduit.

4. Rat sciatic nerve defect repair with nerve conduit containing seed cells

The experimental animals are divided into two groups, namely a control group and an experimental group, the control group uses a nerve conduit without seed cells for treatment, the experimental group uses the nerve stem cells obtained by MB-3 to combine with the nerve conduit for treatment, the nerve conduit is used for connecting the defective sciatic nerves, and the wound surface is sutured. Post-operative observation was continued for 3 months, weekly for animal behavioral testing (SFI score, etc.). After 1 week and 2 weeks after operation, the sciatic nerve of the rat is fixed and is subjected to conventional histochemical staining after being sliced, and the observation shows that the recovery condition of the sciatic nerve of the rat in the experimental group is better than that of the control group, the ethological result is also better than that of the control group, and the result is shown in figure 8. After 3 months of operation, after proper anesthesia, all animals expose sciatic nerves and carry out electrophysiological detection, and the experimental group is superior to the control group, which shows that the neural stem cells after MB-3 treatment can still play the function of repairing nerve injury.

Claims (10)

1. The application of the KAT2A inhibitor in treating spinal cord and sciatic nerve injuries is characterized in that the KAT2A inhibitor is MB-3, and the MB-3 is used for in vitro culture of neural stem cells, so that the proliferation and the balling rate of the neural stem cells are obviously increased, and further the spinal cord and sciatic nerve injuries are treated.
2. 2. The use according to claim 1, wherein the method for ex vivo culture of neural stem cells comprises: using SD pregnant mouse, taking out fetal mouse, separating cerebral cortex, digesting with pancreatin to obtain single cell suspension, and culturing; MB-3 is continuously added during the culture process, and the culture and the passage are continuously carried out until a sufficient number of neural stem cells are obtained.
3. 3. The use according to claim 2, wherein the concentration of MB-3 is 100 μ M.
4. 4. The use of claim 2, wherein the addition of MB-3 is continued during the culturing, the culturing is continued until the stem cell spheres have a diameter of about 150 μm, and then the subculture is continued until a sufficient number of neural stem cells are obtained.
5. 5. The use of claim 2, wherein the primer sequence that detects the change in KAT2A RNA levels is:

   GCN5-sense 5'TCATCGGTGGGATTTGCT 3'；

   GCN5-antisens 5'TACTCGTCGGCGTAGGTG 3'。
6. 6. the use according to claim 1, wherein the neural stem cells cultured in vitro are mixed with the tissue engineering hydrogel for repairing spinal cord injury.
7. 7. The use of claim 6, wherein the tissue-engineered hydrogel is methacrylated silk fibroin.
8. 8. The use of claim 7, wherein the concentration of methacrylated silk fibroin is 5-20%.
9. 9. The use of claim 1, wherein the neural stem cells are cultured on a tissue-engineered nerve conduit and then transplanted for injury repair of sciatic nerve.
10. 10. The use of claim 9, wherein the step of constructing a nerve conduit is: filtering 10% methylacryloyl acylated gelatin solution for sterilization, pouring into a catheter mould to make the inner diameter and the outer diameter of the catheter respectively 2mm and 4mm, freezing at-80 ℃ for 24h after ultraviolet irradiation curing, defrosting and demoulding after freeze-drying, and then co-culturing the neural stem cells and the catheter.

Images