Method for inducing and differentiating Schwann cells by utilizing olfactory ensheathing cells in cooperation with mesenchymal stem cells

Abstract

The invention discloses a method for inducing and differentiating Schwann cells by using olfactory ensheathing cells and mesenchymal stem cells. The method comprises the following steps: mixing adipose-derived mesenchymal stem cells and olfactory ensheathing cells for culturing, and inducing to obtain the Schwann cells. According to the method, the adipose-derived stem cells are differentiated into the Schwann cells through induction culture, the differentiation speed is high, and the functional Schwann cells with the remarkably increased number can be obtained.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of stem cell induction, in particular to a method for inducing adipose-derived stem cells to differentiate into Schwann cells. BACKGROUND

[0002] Adipose-derived stem cells (ADSCs) isolated from human adipose tissue can secrete various growth factors and have the ability of multi-directional differentiation, so ADSCs have become a research hotspot of seed cells in recent years. It has been reported that adipose-derived stem cells were combined with human acellular dermal matrix and transplanted into the skin defect of a nude mouse, and it was found that the implanted cells differentiated into vascular endothelial cells, fibroblasts and epithelial cells, and could effectively promote wound healing. It has also been reported that a method for inducing ADSCs to differentiate into fibroblasts in vitro was given (see CN104805051A).

[0003] Xie et al. reported a method for inducing ADSCs to differentiate into Schwann cells in 2017 (Efficient generation of functional Schwann cells from adipose-derived stem cells in defined conditions, Cell Cycle, 16:9, 841-851), which created favorable conditions for the application of ADSCs in the repair of the nervous system, but it had the disadvantages of complicated induction process and excessive consumption of factors, and the neural function of the differentiated cells was not certain. In addition, there have been reports of inducing differentiation by co-culturing with umbilical cord mesenchymal stem cells, but Schwann cells have not been differentiated (see CN113444689A).

[0004] Olfactory ensheathing cells (OECs) are a kind of glial cells that are functionally between Schwann cells and oligodendrocytes, and have the functions of neurotrophic, inhibiting glial proliferation and scar formation, and sheathing. Although olfactory ensheathing cells provide a suitable microenvironment for axon growth and have strong migration properties, they are one of the ideal candidate cells for promoting central nervous system regeneration, but there have been no reports of using them to induce adipose-derived mesenchymal stem cells to differentiate into Schwann cells. SUMMARY

[0005] The purpose of the present application is to provide a method for inducing mesenchymal stem cells to differentiate into Schwann cells by using olfactory ensheathing cells, which can efficiently induce adipose-derived mesenchymal stem cells to differentiate into Schwann cells with good activity.

[0006] To achieve the above object, the present application adopts the following technical solutions: In a first aspect, an inducing culture method of Schwann cells is provided, comprising the following steps: The adipose-derived mesenchymal stem cells and the olfactory ensheathing cells are mixed at a ratio of 1-5:1 (cell number ratio) and then are co-cultured for 1-2 weeks.

[0007] Preferably, the adipose-derived mesenchymal stem cells are selected from the group consisting of ex vivo cultured cell clones of passages 3-5 (i.e. adipose-derived mesenchymal stem cells having good adipogenic, osteogenic and neuronogenic induction efficiency).

[0008] Preferably, the mixing ratio (cell number ratio) of the adipose-derived mesenchymal stem cells and the olfactory ensheathing cells is 1-3:1 (e.g. 2:1).

[0009] In a second aspect, a nerve injury repair agent is provided, which comprises adipose-derived mesenchymal stem cells and olfactory ensheathing cells, and the mixing ratio (cell number ratio) of the adipose-derived mesenchymal stem cells and the olfactory ensheathing cells is 1-5:1.

[0010] Preferably, the mixing ratio (cell number ratio) of the adipose-derived mesenchymal stem cells and the olfactory ensheathing cells is 1-3:1 (e.g. 2:1).

[0011] Preferably, the dosage form of the repair agent includes but is not limited to injection.

[0012] Preferably, the repair agent is prepared by mixing 1×10 6 cells / mL-2×10 6 cells / mL adipose-derived mesenchymal stem cell suspension with 1×10 6 cells / mL-6×10 6 cells / mL olfactory ensheathing cell suspension.

[0013] In a third aspect, a use of a biological product in the preparation of a medicament for repairing nerve injury is provided, wherein the biological product comprises adipose-derived mesenchymal stem cells and olfactory ensheathing cells, and the mixing ratio (cell number ratio) of the adipose-derived mesenchymal stem cells and the olfactory ensheathing cells is 1-5:1.

[0014] Preferably, the mixing ratio (cell number ratio) of the adipose-derived mesenchymal stem cells and the olfactory ensheathing cells is 1-3:1 (e.g. 2:1).

[0015] Preferably, the nerve is selected from the group consisting of peripheral nerves.

[0016] The beneficial effects of the present application are embodied in: The method for mixing and culturing the adipose tissue-derived mesenchymal stem cells and the olfactory ensheathing cells provided by the application not only realizes the induction and differentiation of the adipose tissue-derived mesenchymal stem cells into Schwann cells, but also has a higher differentiation speed than the existing induction methods (for example, the induction method combined with the supernatant of the olfactory ensheathing cells), so that the Schwann cells with a significantly increased quantity and good functions can be obtained. The application not only improves the induction efficiency of the Schwann cells, but also obtains the active ingredients with a strong nerve injury repair effect through the mixed culture, thereby providing a new way for the functional product development of related biological products. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 It is a cell immunohistochemical staining photo.

[0018] Figure 2 It is a footprint measurement schematic diagram; in the diagram, TOF: four trains of stimulation, this mode induces muscle block recovery.

[0019] Figure 3 It is a frozen section immunofluorescence staining photo. DETAILED DESCRIPTION

[0020] The application will be further described in detail below in combination with the drawings and examples, and the examples are used to explain the application, but not to limit the protection scope of the application.

[0021] (I) Acquisition of rat olfactory ensheathing cells and rat adipose tissue-derived mesenchymal stem cells 1.1 Isolation, culture, purification and identification of rat olfactory ensheathing cells Under sterile conditions, the outer two layers of the rat olfactory bulb were taken, and a cell suspension was prepared by trypsin digestion method. The olfactory ensheathing cells were purified by using the delayed differential adhesion method combined with the cytosine arabinoside inhibitor method, and were identified by detecting the nerve growth factor receptor (p75NGFR) and the glial fibrillary acidic protein (GFAP) antibody. The specific description is as follows.

[0022] 1) Primary isolation and culture of olfactory ensheathing cells Newborn (2-3 months old) SD rats were selected, their skin was disinfected by immersion in 75% alcohol, and they were aseptically decapitated to expose and remove the olfactory bulb (keeping it as intact as possible). The bulb was placed in a culture dish containing D-Hanks (Sigma), and the meninges on the surface of the olfactory bulb were removed. The olfactory bulb was directly torn open with microdissecting forceps, the central portion was removed, and the olfactory nerve layer and granular layer were preserved. The separated tissue fragments were transferred to another test tube, washed twice with D-Hanks, and digested with 0.25% trypsin (Sigma) at 37°C for 25 min. The trypsin-digested tissue fragments were transferred to complete culture medium (Sigma), and DF / 12 medium (Sigma) containing 10% FCS (fetal bovine serum) was added. The tissue fragments were gently pipetted to form a cell suspension. The concentration of DF / 12 medium containing 10% FCS was adjusted to 1×10⁻⁶. 6 ~1.5×10 6 Cells / mL, then inoculated into 25 cm² plates coated with poly-L-lysine. 2 Incubate in a culture flask at 37°C with 5% CO2.

[0023] 2) Purification of olfactory ensheathing cells After 3 days of cell culture, the medium was replaced with fresh complete culture medium, and non-adherent cells and cell debris were removed. Cytarabine (Ara-c, final concentration 5 μmol / L) was added to the complete culture medium and incubated for 48 hours to inhibit rapidly dividing cells such as fibroblasts. The cells were then washed twice with complete culture medium (to remove Ara-c) and cultured again. When the cells reached 85%–90% confluence, differential adhesion was performed for further purification, and the culture supernatant was collected. After centrifugation, a portion of the cells was aliquoted and stored at -80°C for later use, while the remaining portion was seeded onto culture flasks or slides for subsequent experiments.

[0024] 3) Immunohistochemical identification of olfactory ensheathing cells Cells cultured for 6 days were fixed with 4% paraformaldehyde for 20-30 min, washed three times with 0.01M PBS for 5 min each time, and then incubated overnight at 4°C with either 1:200 diluted rabbit anti-nerve growth factor receptor (p75NGFR) antibody or glial fibrillary acidic protein (GFAP) antibody (Abcam) diluted in the same proportion. The cells were then washed three times with 0.01M PBS for 5 min each time, and then incubated for 60 min with 60 min with 0.01M PBS for 5 min each time. Nuclear counterstaining was performed with DIPA and incubated for 10 min with 10 min with 0.01M PBS for 5 min each time to terminate the colorimetric reaction. The cells were mounted with 50% glycerol. A blank control was also provided (no primary antibody was added, all other steps were the same).

[0025] Results see Figure 1 , step 2 separation and purification of the cells have olfactory ensheathing cell-specific nerve growth factor receptor (p75NGFR) and glial fibrillary acidic protein (GFAP) antibody.

[0026] 1.2 Isolation, culture, passage and identification of rat adipose tissue-derived mesenchymal stem cells The rat bilateral inguinal fat tissue was isolated, and the adipose tissue-derived mesenchymal stem cells were isolated and purified by collagenase digestion method. The markers such as CD44 + , CD90 + , CD34 - and CD45 - were detected by flow cytometry, and the identification was carried out by adipogenic induction and osteogenic induction. The specific description is as follows.

[0027] 1) Acquisition of adipose tissue Select 4-6 week old SD rats and anesthetize with 0.1% sodium pentobarbital. Under sterile conditions, remove the bilateral inguinal fat tissue and remove the fibrous components and capillaries under a 10x microscope. After washing with D-Hanks, use ophthalmic scissors to cut the fat tissue.

[0028] 2) Primary isolation and culture of adipose tissue-derived mesenchymal stem cells Place the cut fat tissue in a digestion bottle containing 0.1% collagenase type I (Invitrogen) and digest at 37°C for 30 min. Filter the digestion solution with a 70 μm stainless steel mesh, and continue to digest and filter the undigested tissue pieces. Add an equal volume of DMEM medium (Invitrogen, containing 10% fetal bovine serum, penicillin 100 U / mL, streptomycin 1 mg / mL) to neutralize and dilute the digestion, then centrifuge at 1500 rpm for 7 min. Discard the supernatant, resuspend the cells in a culture flask with DMEM medium (containing 10% fetal bovine serum, penicillin 100 U / mL, streptomycin 1 mg / mL), and culture in a 5% CO2, 37°C, saturated humidity incubator. Change the liquid after 24 hours to remove the unattached cells. Change the liquid every 2-3 days, and then process after the cells have fused into a monolayer.

[0029] 3) Passage of adipose tissue-derived mesenchymal stem cells When the cells grow to 80%~90%, they are subcultured. Before subculture, the cells are washed with a small amount of D-Hanks for 2~3 times to remove the residual medium. The cells are digested with 0.25% trypsin and 0.04% EDTA at 37°C. When most of the cells retract and become round, the digestion is stopped by adding DMEM medium containing 10% FBS. The cells are centrifuged, and the supernatant is discarded. The cells are resuspended in DMEM complete medium and subcultured in another culture bottle at a ratio of 1:2~1:3 (cell density ratio). The medium is changed every 2~3 days.

[0030] 4) Identification of adipose tissue-derived mesenchymal stem cells a) The third generation cells that have reached 90% confluence are digested with trypsin / EDTA into a single cell suspension, washed with PBS for 3 times, counted by a hemocytometer, and adjusted to a concentration of 1×10 6 Anti-CD90-PE, Anti-CD44-FITC, Anti-CD45-PE, Anti-CD34-FITC (Abeam) are added, and the mixture is incubated at 4°C for 30 min. The unlabelled antibodies are washed away with PBS, and the cells are fixed with 1% paraformaldehyde. The positive expression rate of the corresponding labelled antigens in the sample is analyzed by a flow cytometer. The results show that the positive rate is more than 80%.

[0031] b) Adipogenic induction of adipose tissue-derived mesenchymal stem cells The third generation cells are plated in 24-well plates (8 wells are plated). When the cells have reached confluence, the cells in 6 wells are cultured in adipogenic induction medium at 37°C, 5% CO2, and saturated humidity. The cells in the remaining 2 wells are cultured in DMEM medium containing 10% FBS as a control group. The medium is changed every 2~3 days. After 7 days of culture, the morphological changes of the cells in each group are observed under a light microscope. After 4 weeks of culture, the cells are washed with PBS once. Von Kossa staining is performed according to the instructions of the alkaline phosphatase staining kit, and the cells are counterstained with nuclear fast red reagent. The results are observed under a microscope. The adipogenic induction medium is composed of DMEM complete medium, 0.1 μmol / L dexamethasone, 50 μmol / L 2-phosphoascorbic acid, and 10 mmol / L β-glycerophosphate.

[0032] The results show that the induction efficiency of the experimental group is significantly higher than that of the control group, and the adipogenic induction experiment is successful.

[0033] c) Osteogenic induction of adipose tissue-derived mesenchymal stem cells The fifth generation cells are digested with 0.25% trypsin, and the cell concentration is adjusted to 5×10 5The cells were adjusted to a concentration of 5 x 105cells / mL and plated in 24-well plates (8 wells per plate). When the cells were completely fused, 6 wells of cells were taken and placed in an osteogenic induction medium and cultured in a 37°C, 5% CO2, saturated humidity incubator, serving as an experimental group. The remaining 2 wells of cells were cultured in a DMEM medium containing 10% FBS, serving as a control group. The medium was changed every 2-3 days, and after 5 days of culture, the morphological changes of the cells in each group were observed under a light microscope. After 2 weeks of culture, the cells were stained with an oil red O solution (3 parts by volume of 3.75% oil red O in isopropyl alcohol and 2 parts by volume of distilled water) for 5 minutes and observed under a microscope. The osteogenic induction medium was formulated as follows: DMEM complete culture medium + 0.1 μmol / L dexamethasone + 10 μmol / L insulin + 0.5 mmol / L 3-isobutyl-1-methyl xanthine + 0.2 mmol / L indomethacin.

[0034] The results show that the induction efficiency of the experimental group is significantly higher than that of the control group, and the osteogenic induction experiment is successful.

[0035] (B) Multidirectional differentiation ability of adipose-derived mesenchymal stem cells In addition to the adipogenic and osteogenic induction experiments, the adipose-derived mesenchymal stem cells isolated were also subjected to a neural-like cell induction experiment. The details are as follows.

[0036] The 3rd passage cells were digested with 0.25% trypsin, and the cell concentration was adjusted to 5 x 105cells / mL and plated in 24-well plates (8 wells per plate). When the cells were completely fused, 6 wells of cells were taken and placed in an osteogenic induction medium and cultured in a 37°C, 5% CO2, saturated humidity incubator, serving as an experimental group. The remaining 2 wells of cells were cultured in a DMEM medium containing 10% FBS, serving as a control group. The medium was changed every 2-3 days, and after 5 days of culture, the morphological changes of the cells in each group were observed under a light microscope. After 2 weeks of culture, the cells were stained with an oil red O solution (3 parts by volume of 3.75% oil red O in isopropyl alcohol and 2 parts by volume of distilled water) for 5 minutes and observed under a microscope. The osteogenic induction medium was formulated as follows: DMEM complete culture medium + 0.1 μmol / L dexamethasone + 10 μmol / L insulin + 0.5 mmol / L 3-isobutyl-1-methyl xanthine + 0.2 mmol / L indomethacin. 4 5 The cells were adjusted to a concentration of 5 x 105cells / mL and plated in 24-well plates (8 wells per plate). When the cells were completely fused, 6 wells of cells were taken and placed in an osteogenic induction medium and cultured in a 37°C, 5% CO2, saturated humidity incubator, serving as an experimental group. The remaining 2 wells of cells were cultured in a DMEM medium containing 10% FBS, serving as a control group. The medium was changed every 2-3 days, and after 5 days of culture, the morphological changes of the cells in each group were observed under a light microscope. After 2 weeks of culture, the cells were stained with an oil red O solution (3 parts by volume of 3.75% oil red O in isopropyl alcohol and 2 parts by volume of distilled water) for 5 minutes and observed under a microscope. The osteogenic induction medium was formulated as follows: DMEM complete culture medium + 0.1 μmol / L dexamethasone + 10 μmol / L insulin + 0.5 mmol / L 3-isobutyl-1-methyl xanthine + 0.2 mmol / L indomethacin. 2 Olfactory ensheathing cells were co-cultured with the cells (neuronal cell induction medium, 37°C, 5% CO2, saturated humidity incubator), serving as an experimental group; the remaining 2 wells of cells were cultured in a DMEM medium containing 10% FBS, serving as a control group. The medium was changed every 2-3 days, and after 2 weeks of culture, the cells were observed under a microscope, and the cell RNA was extracted for Tuj-1, GFAP, and MBP mRNA RT-PCR analysis and identification. The neuronal cell induction medium was formulated as follows: DMEM / F12 (Invitrogen) + 2% FCS + 5 μM retinoic acid (RA) + 1% G5 + 50 μg / mL penicillin + 50 μg / mL streptomycin.

[0037] The results show that the above genes are highly expressed in the experimental group, significantly higher than in the control group, and the neural-like cell induction experiment is successful. Therefore, the adipose-derived mesenchymal stem cells isolated have a multidirectional differentiation ability.

[0038] (Three) Olfactory ensheathing cells cooperated with adipose-derived mesenchymal stem cells to induce differentiation into Schwann cells ​The cultured adipose-derived mesenchymal stem cells were induced to differentiate into Schwann cells (using the method of mixed culture with olfactory ensheathing cells), and compared with the method of traditional cytokine induction combined with olfactory ensheathing cell supernatant induction (Xie et al.). The morphology of the differentiated Schwann cells was observed under a light microscope, and the transformation efficiency of the Schwann cells was analyzed by immunocytochemical staining. The specific description is as follows.

[0039] 3.1 Microenvironment protein array analysis of olfactory ensheathing cell secreted activity The adipose-derived mesenchymal stem cells (passage 3) were divided into five groups: simple adipose-derived mesenchymal stem cell group; Transwell olfactory ensheathing cell group (without olfactory ensheathing cells but with secreted trophic factors); olfactory ensheathing cell mixed culture (OECs:ADSCs=1:5, cell number ratio) group; olfactory ensheathing cell mixed culture (OECs:ADSCs=1:2, cell number ratio) group; and olfactory ensheathing cell mixed culture (OECs:ADSCs=1:1, cell number ratio) group.

[0040] Culture conditions: 5% CO2, 37°C; DMEM culture medium containing 10% FCS; cultured to logarithmic growth phase.

[0041] The changes of neurotrophic and supporting factors in the microenvironment of cell supernatant, such as PDGF, NPY, BDNF, NGF, GDNF, NT-3, NT-4, and CNTF, were analyzed using protein chip analysis technology (using Ray. Biotech products), and the expression of molecules related to cell adhesion and axon growth, such as L1(100), PSA-N-CAM, N-CAM, laminin, fibronectin, nexin, and S100, which regulate the elongation of olfactory axons, was analyzed.

[0042] Through protein array analysis, the optimal cell mixing ratio was screened as 1:2 (OECs:ADSCs).

[0043] 3.2 Induction of adipose-derived mesenchymal stem cells to differentiate into Schwann cells 3.2.1 Traditional cytokine induction After the adipose tissue-derived mesenchymal stem cells adhered, the culture solution was removed, and the cells were washed with D-Hanks for 2-3 times. Then, 1 mmol / L of β-mercaptoethanol (BME, Sigma) was added, and the cells were cultured in a 37°C, 5% CO2, and saturated humidity incubator for 24 hours. After 24 hours, the liquid was removed and washed with D-Hanks for 2 times. The cells were cultured in DMEM culture solution containing 35 ng / mL all-trans retinoic acid (ATRA, Sigma) and 10% fetal bovine serum for 3 days. After 3 days, the culture solution was discarded, and the cells were washed with D-Hanks. Then, the complex induction factors were added in the complete culture solution. The complex induction factors included 5 mmol / L forskolin (FSK, Sigma), 10 ng / mL recombinant human basic fibroblast growth factor (bFGF, Sigma), 5 ng / mL recombinant platelet-derived factor (PDGF-AA, Sigma), and 200 ng / mL recombinant human heregulin-β (HRG-β, Sigma). The cells were cultured for 7 days under the action of the above-mentioned complex induction factors, and the cells were not subcultured during the period. Under the observation of an inverted phase contrast microscope, the typical Schwann cell characteristics such as spindle shape were found.

[0044] 3.2.2 Combined induction by olfactory ensheathing cell supernatant The adipose tissue-derived mesenchymal stem cells were inoculated into 6-well or 24-well plates, respectively. After the cells adhered, the culture solution was removed, and the cells were washed with D-Hanks for 2-3 times. Then, 1 mmol / L of β-mercaptoethanol (BME, Sigma) was added, and the cells were cultured in a 37°C, 5% CO2, and saturated humidity incubator for 24 hours. After 24 hours, the liquid was removed and washed with D-Hanks for 2 times. The cells were cultured in DMEM culture solution containing 35 ng / mL all-trans retinoic acid (ATRA) and 10% fetal bovine serum for 3 days. After 3 days, the culture solution was discarded, and 5 mmol / L forskolin-containing DMEM culture solution was added. The Transwell chamber in which the olfactory ensheathing cells were cultured was moved to the adipose tissue-derived mesenchymal stem cell culture plate, and the cells were continuously cultured for 1-2 weeks. Under the observation of an inverted phase contrast microscope, the typical Schwann cell characteristics such as spindle shape were found.

[0045] 3.2.3 Induction by mixed culture of olfactory ensheathing cells The purified olfactory ensheathing cells were inoculated into 6-well or 24-well plates, respectively, and were used for co-culture with the adipose tissue-derived mesenchymal stem cells after being cultured for 3-5 days.

[0046] Example of co-culture: After 3 days, the adipose tissue-derived mesenchymal stem cells and the olfactory ensheathing cells were digested with 0.125% and 0.02% trypsin, respectively, and were prepared into single-cell suspensions. Then, the adipose tissue-derived mesenchymal stem cells were inoculated into the 6-well plates at a concentration of 5×10 4 / cm 2The density of the cells was inoculated into the culture plate containing olfactory ensheathing cells, i.e. mixed with olfactory ensheathing cells at an optimal cell mixing ratio of 1:2 (OECs:ADSCs), cultured for 1-2 weeks (5% CO2, 37°C sterile incubator), and then observed for changes in cell morphology under an inverted phase contrast microscope. Typical Schwann cell characteristics such as fusiform cells were found.

[0047] 3.3 Immunohistochemical identification of cells after induction After the above three methods of induction, the cell climbing sheet was taken out, the culture solution was aspirated, and PBS solution was washed 3 times. After being fixed with 4% paraformaldehyde for 15 min, the cells were washed with PBS solution 3 times for 2-3 min each time, 0.01% Triton-X100 was added, and the cells were incubated at room temperature for 30 min. After blocking with goat serum for 30 min at room temperature, the cells were washed without washing, and mouse anti-rat GFAP (1:100, Abeam) and rabbit anti-rat S-100 (1:100, Abeam) were added. The control group was added with PBS solution. The cells were incubated in a wet box at 4°C overnight. The cells were washed with PBS solution 3 times for 3 min each time, and then the PBS solution was removed. Fluorescent (Cy3) labeled goat anti-mouse IgG and fluorescent labeled (FITC) goat anti-rabbit IgG were added as secondary antibodies, and the cells were incubated at 37°C in the dark for 60 min. The cells were washed with PBS solution 4 times for 5 min each time, and then the PBS solution was removed. 10 mg / L DAPI was added, and the cells were incubated at 37°C in the dark for 30 min. The cells were washed with PBS solution 2 times for 3 min each time, and then mounted with glycerol. The cells were observed and photographed under a fluorescent inverted phase contrast microscope. Ten non-overlapping fields were randomly counted, and the ratio of positive cells (i.e. induced Schwann cells, identified by carrying both GFAP and S-100) in each group to the total number of counted cells in the field was calculated.

[0048] The results showed that the positive cell ratio induced by olfactory ensheathing cell mixed culture was 50%-80%, the positive cell ratio induced by combined olfactory ensheathing cell supernatant was 30%-40%, and the positive cell ratio induced by traditional cytokines was 20-40%.

[0049] (Four) Olfactory ensheathing cells combined with adipose mesenchymal stem cells for repairing peripheral nerve injury around electric burn 4.1 Establishment of rat electric burn peripheral nerve injury model Taking the rat sciatic nerve electric burn model as an example, the process was established as follows: Rats were injected intraperitoneally with 3% sodium pentobarbital (30 mg / kg); after successful anesthesia, the rats were placed in a prone position, and the limbs were fixed with the limbs spread out. The right hip and the back of the thigh were shaved and prepared for skin, and then iodine and alcohol were used for disinfection, and a disposable hole towel was laid. A longitudinal incision was made in the middle of the thigh, and the skin, fascia, and muscle were sequentially incut, and the muscle margins on both sides were sutured to the skin for automatic retraction. The sciatic nerve was exposed, and the sciatic nerve was carefully dissected and isolated. The sciatic nerve of one side of the rat was separated for about 1 cm, and the voltage was 5 kv, the action time was 1 second, and the pathological section proved the degree of injury. All test animals were injected intraperitoneally with bromodeoxyuridine (BrdU, a cell division S phase marker, used to evaluate the number of new neurons) at 0, 1, 2, 3, 4, 5, 6, and 7 days after injury, with a dose of 100 mg / kg.

[0050] 4.2 Olfactory ensheathing cells combined with adipose-derived mesenchymal stem cells to repair rat electrical burn nerve injury 1) Animal grouping 65 healthy SD rats, male and female, weighing 200-250 g, were randomly divided into groups: normal control group (5 rats); negative treatment group (normal saline), adipose-derived mesenchymal stem cell group, simple olfactory ensheathing cell group, and olfactory ensheathing cell combined with adipose-derived mesenchymal stem cell group (15 rats in each treatment group).

[0051] 2) Treatment experiment a) Cell preparation When the 3rd generation of ADSCs and OECs reached 80%-90% confluence, they were digested with 0.125% trypsin, washed with PBS, and adjusted to 1×10 6 / mL, respectively, for standby.

[0052] b) Injection method in the treatment experiment 2 μL of normal saline, single cell suspension, and mixed cell suspension (at the optimal cell mixing ratio described above) were injected into the endoneurium 2 mm away from the denervation end (avoiding injury to the nerve fibers) at intervals of 2 mm.

[0053] 3) Detection indicators for rat sciatic nerve repair a) Gross observation Postoperative limb paralysis, gait, ability to stand stably, wound healing, ulceration and healing of the plantar surface, and the morphology, luster, texture of the repaired nerve and adhesion to the surrounding tissue were observed.

[0054] b) Hot stimulus withdrawal reflex (withdrawal reflex, WRL) experiment The withdrawal reflex time was defined as the time from the rat's hind paw contacting the hot plate to the withdrawal. The rat was wrapped with a sterile cloth, and the left hind limb was exposed. The rat's left hind paw was placed on the hot plate at 56°C. The time from the paw contacting the hot plate to the withdrawal was measured with a stopwatch. The measurement was repeated three times at intervals of 2 min, and the average value was taken.

[0055] c) Electrophysiological detection At 21 days after the operation, all rats were subjected to electrophysiological detection at room temperature. The rats were weighed, and a compound anesthetic was injected intraperitoneally. After successful anesthesia, the sciatic nerve was exposed, and a glass needle was used to carefully separate the sciatic nerve. The compound muscle actin potentials (CMAPs) were recorded according to the method of Suzuki. The recording electrode was inserted into the gastrocnemius muscle belly, the interference electrode was placed on the knee skin of the rat, and the stimulating electrode was placed on the sciatic nerve trunk near and far from the burn site in turn. The nerve was stimulated, and the CMAPs were recorded to measure the amplitude and latency of the CMAPs. In the same way, the amplitude and latency of the CMAPs of the normal side were recorded. The amplitude of the CMAPs is proportional to the number of nerve fibers innervating the target muscle, so detecting the CMAPs provides an important parameter for the recovery of peripheral nerve conduction function: Recovery index = maximum amplitude of CMAPs on the operation side / maximum amplitude of CMAPs on the normal side d) Sciatic nerve function index (SFI) determination A homemade rat footprint walking box (12 cm wide and 100 cm long) was used. White paper was placed at the bottom of the box. The rats in each group were placed at one end of the walking box after their feet were dipped in carbon ink. The rats walked to the other end on their own. The experimental injury side foot (Sciatic, E) and the normal side foot (Normal, N) left footprints on the white paper. Three to four good footprints were selected (see Figure 2 ). Three variables were measured for the footprints on the normal side and the experimental injury side, respectively: footprint length (PL), which is the distance from the heel to the tip; toes preading (TS), which is the distance between the first and fifth toes; and intermediate toes preading (IT), which is the distance between the second and fourth toes. The measured values of the above three variables were substituted into the Bain formula to calculate the SFI. The recovery rate of the SFI was calculated with SFI = 0 as the normal value and SFI = 100 as the indicator of complete nerve disconnection.

[0056]

[0057] e) Tibilais anterior muscle wet weight ratio determination After the rats in each group were measured by neurophysiology, the tibialis anterior muscles of the healthy (left) and diseased (right) sides were removed, and the wet weights of the tibialis anterior muscles were measured on an electronic balance with an accuracy of one ten-thousandth. Then, the wet weight ratio of the tibialis anterior muscle was calculated: Tibialis anterior muscle wet weight ratio = diseased tibialis anterior muscle wet weight / healthy tibialis anterior muscle wet weight x 100% f) Histological observation of the number of nerve fibers, nerve fiber myelin sheath and axon The middle section of the removed nerve graft was fixed with 2.5% glutaraldehyde, then placed in a 4% osmium tetroxide solution for 2 hours at room temperature, dehydrated with gradient alcohol, replaced with acetone, embedded with epoxy resin, made into semi-thin sections, stained with 1% toluidine blue solution, and observed under a light microscope. Ultra-thin sections were also prepared, stained with acetic acid bichromate and lead acetate, and observed under a JEM-1200EX transmission electron microscope for ultrastructure observation. The number of regenerated nerve fibers, nerve fiber myelin sheath thickness and axon diameter were analyzed using a MetaMorph / DPl0 / BX41 image analysis system.

[0058] The results showed that the repair effect of the olfactory ensheathing cell and adipose mesenchymal stem cell mixed group was significantly better than that of the other treatment groups, for example, the recovery rate of SFI of the olfactory ensheathing cell and adipose mesenchymal stem cell mixed group was 60%, the recovery rate of SFI of the negative treatment group was 5%, the recovery rate of SFI of the adipose mesenchymal stem cell group was 30%, and the recovery rate of SFI of the simple olfactory ensheathing cell group was 30%.

[0059] 4) Preparation of frozen sections for immunofluorescence tracing of labeled transplanted cells From each group of rats injected with transplanted cells, 4 rats were randomly selected to remove the nerve fibers in the cells of the rats, fixed with 4% paraformaldehyde overnight, washed with PBS solution for 3 times the next day, then placed in a 30% sucrose solution overnight, then embedded with OCT, made into frozen sections (8 μm thick), and placed on glass slides coated with polylysine. Fixed with cold acetone for 10 min, washed with PBS solution for 5 min, perforated with 0.5% Triton X-100 for 15 min, washed with PBS solution for 2 times, each for 5 min, blocked with 1% BSA for 30 min, the primary antibody was anti-S-100 antibody (1:150 dilution, Abeam), incubated overnight after adding the primary antibody, washed with PBS solution for 5 min the next day, added with secondary antibody TRITC (1:200 dilution, Abeam) solution, incubated for 40 min, washed with PBS solution for 5 min, stained with DAPI for 2-5 min, mounted, and observed under a microscope. Figure 3 The S-100 fluorescence was red, and the adipose mesenchymal stem cells were green. According to the observation under the microscope, the conversion ratio (the conversion ratio refers to the ratio of the number of Schwann cells differentiated from the adipose mesenchymal stem cells in the transplanted cells to the total number of adipose mesenchymal stem cells transplanted) was calculated.

[0060] The results showed that the conversion ratio of the group of olfactory ensheathing cells mixed with adipose tissue-derived mesenchymal stem cells was 60%, the conversion ratio of the group of adipose tissue-derived mesenchymal stem cells was 30%, and the conversion ratio of the group of olfactory ensheathing cells was 30%. Obviously, the conversion ratio of the group of olfactory ensheathing cells mixed with adipose tissue-derived mesenchymal stem cells was higher than that of other groups of non-mixed cells.

Claims

1. A method for inducing culture of Schwann cells, characterized by: The method comprises the following steps: The adipose-derived mesenchymal stem cells and the olfactory ensheathing cells are mixed at a ratio of 1-5:1 and then are co-cultured for 1-2 weeks.

2. The method for inducing and culturing Schwann cells according to claim 1, characterized in that: The adipose-derived mesenchymal stem cells are selected from the group consisting of in-vitro cultured cell clones of passages 3-5.

3. The method for inducing and culturing Schwann cells according to claim 1, characterized in that: The mixing ratio of the adipose-derived mesenchymal stem cells and the olfactory ensheathing cells is 1-3:

1.

4. A nerve injury repair agent, characterized by: The repairing agent comprises adipose-derived mesenchymal stem cells and olfactory ensheathing cells, and the mixing ratio of the adipose-derived mesenchymal stem cells and the olfactory ensheathing cells is 1-5:

1.

5. The nerve injury repair agent according to claim 4, characterized in that: The mixing ratio of the adipose-derived mesenchymal stem cells and the olfactory ensheathing cells is 1-3:

1.

6. The nerve injury repair agent of claim 4, wherein: The dosage form of the repairing agent is injection.

7. The nerve injury repair agent of claim 4, wherein: The repair agent is mixed from 1-2 x 10 6 cells / mL of a mesenchymal stem cell suspension and 1-6 x 10 6 cells / mL of an olfactory ensheathing cell suspension.

8. Use of a biological product for the preparation of a medicament for the repair of a nerve injury, characterized in that: The biological product comprises adipose-derived mesenchymal stem cells and olfactory ensheathing cells, and the mixing ratio of the adipose-derived mesenchymal stem cells and the olfactory ensheathing cells is 1-5:

1.

9. Use according to claim 8, characterized in that: The mixing ratio of the adipose-derived mesenchymal stem cells and the olfactory ensheathing cells is 1-3:

1.

10. Use according to claim 8, characterized in that: The nerve is selected from the group consisting of peripheral nerves.

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