Method for promoting neural differentiation of mesenchymal stem cells by regulating target genes through exosomal miRNA

Abstract

The present invention discloses a method for promoting neural differentiation of mesenchymal stem cells by regulating target genes through exosomal miRNA, and belongs to the field of biotechnology. The method for promoting neural differentiation of mesenchymal stem cells by regulating target genes through exosomal miRNA includes the following steps: S1: extracting mesenchymal stem cells; S2: freezing and storing the obtained mesenchymal stem cells; S3: taking out the non-destructive sampling cryopreservation tube; S4: culturing with MSC culture medium until the culture dish is covered; S5: isolating mononuclear cells; S6: obtaining neural stem cell-like cells; S7: obtaining differentiated neural cells. The present invention solves the problem that the mesenchymal stem cells cultured in the prior art will reduce the immunoregulatory ability of mesenchymal stem cells and affect their application in the treatment of immune-related diseases. The present invention can promote cell proliferation and differentiation and improve the immunoregulatory ability of mesenchymal stem cells.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and specifically to a method for promoting the neural differentiation of mesenchymal stem cells by regulating target genes through exosomal miRNAs. Background Art

[0002] Mesenchymal stem cells are a type of pluripotent stem cells, which have all the commonalities of stem cells, namely self-renewal and multi-directional differentiation ability. They are also the most widely used in clinical applications. When combined with hematopoietic stem cells, the success rate of transplantation can be increased and hematopoietic reconstruction can be accelerated. When patients receive high-dose chemotherapy, injecting mesenchymal stem cells and hematopoietic stem cells together can significantly accelerate the recovery time of blood cells in patients, and it is safe without adverse reactions.

[0003] Chinese Patent with publication number CN117363567A discloses a new method for extracting mesenchymal stem cells, including the following steps: Step 1: Collect mesenchymal stem cells; Step 2: Suspend the mesenchymal stem cells in a medium containing high-concentration sheep serum under sterile conditions; Step 3: Transfer the medium to a low-adhesion culture dish; Step 4: Extract mesenchymal stem cells at 37°C for 24 hours; Step 5: Remove non-adherent cells and residual sheep serum, and add serum-free medium; Step 6: Continue to culture mesenchymal stem cells in a low-adhesion culture dish; Step 7: Regularly change the serum-free medium to maintain the growth and proliferation of mesenchymal stem cells. By using bone marrow, umbilical cord blood, umbilical cord tissue, placental tissue, and adipose tissue, the activity of mesenchymal stem cells is ensured, so that the culture survival rate is 30% higher than that of traditional culture methods, and at the same time, the proliferation ability of mesenchymal stem cells is enhanced.

[0004] In the actual use of the above patent, since mesenchymal stem cells play an important role in tissue repair and regeneration, and this role is partially achieved through the exosomes secreted by them, the mesenchymal stem cells cultured by the above method do not utilize exosomes. Therefore, the immunomodulatory ability of mesenchymal stem cells will be reduced, affecting their application in the treatment of immune-related diseases and not meeting the existing needs. For this reason, we propose a method for promoting the neural differentiation of mesenchymal stem cells by regulating target genes through exosomal miRNAs. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for promoting the neural differentiation of mesenchymal stem cells by regulating target genes through exosomal miRNAs, so that the differentiated neural cells have high biological activity, can promote cell proliferation and differentiation, can help mesenchymal stem cells grow and differentiate into the required cell types better, and have an improved immunomodulatory effect, can inhibit inflammatory reactions, promote tissue repair, and participate in the activation and differentiation processes of immune cells, improving the treatment effect, and solving the problems raised in the above background art.

[0006] To achieve the above object, the present invention provides the following technical solutions: A method for promoting the neural differentiation of mesenchymal stem cells by regulating target genes through exosomal miRNA, comprising the following steps:

[0007] S1: Extract mesenchymal stem cells;

[0008] S2: After placing the obtained mesenchymal stem cells into a non-destructive sampling cryopreservation tube, put them into a cryopreservation and recovery box for cryopreservation, and finally transfer them to liquid nitrogen for cryopreservation storage;

[0009] S3: After resuscitating the mesenchymal stem cells using a cryopreservation and recovery box, take out the non-destructive sampling cryopreservation tube and detect the cell viability;

[0010] S4: Take the resuscitated mesenchymal cells with a cell viability greater than 80% for culture, and culture the mesenchymal stem cells in MSC medium until they cover the culture dish;

[0011] S5: After digesting the mesenchymal stem cells on the culture dish, use Percoll separation solution to separate mononuclear cells;

[0012] S6: Modify the cell medium and purify the exosomal miRNA, add the purified exosomal miRNA to the modified cell medium for purification and amplification culture of mesenchymal stem cells for 3 days to obtain neural stem cell-like cells;

[0013] S7: Transfer the neural stem cell-like cells to MesenCult~TM medium and continue to culture for 7 - 14 days to obtain differentiated neural cells.

[0014] Preferably, the biomarkers of the exosomal miRNA include: hsa-miR-652-3p, hsa-miR-30e-5p, hsa-miR-185-5p, and hsa-let-7a-5p.

[0015] Preferably, the extraction of mesenchymal stem cells specifically includes:

[0016] First, collect mesenchymal stem cells, and suspend the mesenchymal stem cells in a medium containing high-concentration sheep serum under sterile conditions;

[0017] Transfer the medium to a low-adhesion culture dish and extract mesenchymal stem cells at 37°C for 24 hours;

[0018] Remove non-adherent cells and residual sheep serum, add serum-free medium, and continue to culture mesenchymal stem cells in a low-adhesion culture dish.

[0019] Preferably, the purification of the exosomal miRNA specifically includes:

[0020] Sample preparation: Transfer samples such as serum into centrifuge tubes, and take the supernatant after centrifugation.

[0021] Magnetic bead preparation: Take out the magnetic beads in the kit, wash them and then add the samples.

[0022] Incubation: Mix the magnetic beads and the samples for incubation.

[0023] Magnetic bead collection: Aggregate the magnetic beads by centrifugation and magnetic stand, and discard the supernatant.

[0024] Exosome elution: Use buffer to elute the exosomes, and the collected eluate is the purified exosomal miRNA solution.

[0025] Preferably, the components of the improved cell culture medium include ginsenoside Rg1, notoginseng polysaccharide, vitamin C, EGF and grape seed extract.

[0026] Preferably, the mesenchymal stem cells are prepared by extracting from one or more of bone marrow, cord blood, umbilical cord tissue, placental tissue and adipose tissue.

[0027] Preferably, the MSC culture medium includes MEM-α basal medium, component A and component B. Among them, component A is composed of the following components: elagolix and thrombospondin, and component B is composed of human platelet lysate.

[0028] Preferably, the volume ratio of elagolix in the basal medium is 0.1%-5%, the volume ratio of thrombospondin in the basal medium is 0.1%-5%, and the volume ratio of platelet lysate in the basal medium is 5%-20%.

[0029] Compared with the prior art, the beneficial effects of the present invention are:

[0030] In the present invention, by purifying exosomal miRNA, impurities and interfering substances can be removed. When differentiating mesenchymal stem cells, adding the purified exosomal miRNA makes the differentiated nerve cells have high biological activity, can promote cell proliferation and differentiation, can help mesenchymal stem cells grow and differentiate into the required cell types better, and has an improved immunomodulatory effect, can inhibit inflammatory reactions, promote tissue repair, and participate in the activation and differentiation processes of immune cells, improving the treatment effect. At the same time, adding the purified exosomal miRNA can maintain biological activity for a long time, and is easy to store and transport, facilitating clinical application. Among them, the intermittent hypoxic atmosphere culture process and the ultrasonic co-culture process can significantly promote differentiation and increase NGF secretion. Description of the Drawings

[0031] Figure 1Schematic diagram of the method for promoting neural differentiation of mesenchymal stem cells by regulating target genes through exosomal miRNAs in the present invention. Detailed implementation manners

[0032] The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention. Example 1

[0033] To solve the problem that existing cultured mesenchymal stem cells do not utilize exosomes, which will reduce the immunomodulatory ability of mesenchymal stem cells and affect their application in the treatment of immune-related diseases, please refer to Figure 1 The following technical solutions are provided in this example:

[0034] A method for promoting neural differentiation of mesenchymal stem cells by regulating target genes through exosomal miRNAs, comprising the following steps:

[0035] S1: Extract mesenchymal stem cells;

[0036] S2: After placing the obtained mesenchymal stem cells into a non-destructive sampling cryopreservation tube, put it into a cryopreservation and recovery box for cryopreservation. The cryopreservation sequence is: cryopreservation in ice water for 2 h, dry ice for 12 h, and finally transfer to liquid nitrogen for storage;

[0037] Cryopreserving mesenchymal stem cells can not only maintain the viability and function of mesenchymal stem cells, but also extend the shelf life of mesenchymal stem cells. Live cells transported in the culture medium have a very short shelf life, while cryopreservation can significantly extend the storage time of stem cells, enabling them to be used when needed. Cryopreservation can also improve the treatment efficiency, and the cryopreserved mesenchymal stem cells can still maintain their immunomodulatory function after thawing.

[0038] S3: After thawing the mesenchymal stem cells using a cryopreservation and recovery box, take out the non-destructive sampling cryopreservation tube and detect the cell viability;

[0039] S4: Take the thawed mesenchymal cells with a cell viability greater than 80% for culture, and culture the mesenchymal stem cells in MSC medium until they cover the culture dish;

[0040] S5: After digesting the mesenchymal stem cells on the culture dish, use Percoll separation solution to separate mononuclear cells;

[0041] S6: Improve the cell culture medium and purify exosomal miRNA. Add the purified exosomal miRNA to the improved cell culture medium for purification and amplification culture of mesenchymal stem cells for 3 days to obtain neural stem cell-like cells;

[0042] When differentiating mesenchymal stem cells, add the purified exosomal miRNA, so that the differentiated nerve cells have high biological activity, can promote cell proliferation and differentiation, can help mesenchymal stem cells grow and differentiate into the required cell types better, and have an improved immunomodulatory effect, can inhibit inflammatory responses, promote tissue repair, and participate in the activation and differentiation processes of immune cells. This immunomodulatory effect helps reduce transplant rejection reactions and improve the safety of cell therapy. As a drug carrier, exosomes have low immunogenicity and good biocompatibility as a drug carrier, can be delivered to the target tissue through multiple in vivo pathways, and as a carrier of drugs or therapeutic factors, can improve the therapeutic effect.

[0043] S7: Transfer the neural stem cell-like cells to MesenCult~TM medium and continue to culture for 7 - 14 days to obtain differentiated nerve cells.

[0044] The biomarker of exosomal miRNA includes: hsa-miR-652-3p.

[0045] Extract mesenchymal stem cells, specifically including:

[0046] Collect mesenchymal stem cells and suspend the mesenchymal stem cells in a medium containing high-concentration fetal bovine serum under sterile conditions. The concentration of fetal bovine serum is 10%;

[0047] Transfer the medium to a low-attachment culture dish and extract mesenchymal stem cells at 37°C for 24 hours;

[0048] Remove non-adherent cells and residual fetal bovine serum, add serum-free medium, and continue to culture mesenchymal stem cells in a low-attachment culture dish, and regularly change the serum-free medium to maintain the growth and proliferation of mesenchymal stem cells.

[0049] Purify exosomal miRNA, specifically including:

[0050] Sample preparation: Transfer samples such as serum into a centrifuge tube, and take the supernatant after centrifugation;

[0051] Magnetic bead preparation: Take out the magnetic beads in the kit, wash them and add the sample;

[0052] Incubation: Mix and incubate the magnetic beads with the sample;

[0053] Magnetic bead collection: Aggregate the magnetic beads by centrifugation and a magnetic stand, and discard the supernatant;

[0054] Exosome elution: Use buffer to elute exosomes, and the collected eluate is the purified exosome miRNA solution. The buffer is phosphate buffer.

[0055] The benefits of purifying exosome miRNA mainly include improving the accuracy and reliability of research, as well as discovering new biomarkers and therapeutic targets. By purifying exosome miRNA, impurities and interfering substances can be removed, making the research results more accurate and reliable. In addition, the purified exosome miRNA can also be used for disease diagnosis and prognosis evaluation, providing new possibilities for clinical applications. In addition, the purified exosome miRNA can also be used as potential biomarkers for early disease diagnosis and prognosis evaluation. For example, the level of serum exosome miR-223 in dementia patients is related to the severity of the disease and can be used as a marker for diagnosing and evaluating disease progression. Similarly, new biomarkers and treatment methods have also been discovered in the research of exosome miRNA in obesity and insulin resistance, and these findings provide new possibilities for the treatment and prevention of diseases.

[0056] The components of the modified cell culture medium include ginsenoside Rg1, notoginseng polysaccharide, vitamin C, EGF, and grape seed extract. These components work together to improve the induction efficiency, and the composition of the culture medium is simple, facilitating preparation and use.

[0057] Among them, the content of ginsenoside Rg1 is 23.08 μg / mL;

[0058] The content of notoginseng polysaccharide is 0.13 μg / ml;

[0059] The content of vitamin C is 3 μg / ml;

[0060] The content of EGF is 0.1 ng / ml;

[0061] The content of grape seed extract is 0.8 μg / ml.

[0062] Mesenchymal stem cells are prepared by extraction from bone marrow.

[0063] The MSC culture medium includes MEM-α basal medium, component A, and component B. Among them, component A consists of the following components: exenatide and thrombospondin, and component B consists of human platelet lysate.

[0064] The volume ratio of exenatide to the basal medium is 5%, the volume ratio of thrombospondin to the basal medium is 5%, and the volume ratio of platelet lysate to the basal medium is 20%. Example 2

[0065] Method for promoting neural differentiation of mesenchymal stem cells by regulating target genes through exosomal miRNA, comprising the following steps:

[0066] S1: Extract mesenchymal stem cells;

[0067] S2: After placing the obtained mesenchymal stem cells into a non-destructive sampling cryopreservation tube, put it into a cryopreservation and recovery box for cryopreservation, and finally transfer it to liquid nitrogen for storage;

[0068] S3: After thawing the mesenchymal stem cells using a cryopreservation and recovery box, take out the non-destructive sampling cryopreservation tube and detect the cell viability;

[0069] S4: Take the thawed mesenchymal cells with a cell viability greater than 80% for culture, and culture the mesenchymal stem cells in MSC medium until they cover the culture dish;

[0070] S5: After digesting the mesenchymal stem cells on the culture dish, use Percoll separation solution to separate mononuclear cells;

[0071] S6: Modify the cell medium, add exosomal miRNA to the modified cell medium for purification and amplification culture of mesenchymal stem cells for 3 days to obtain neural stem cell-like cells;

[0072] S7: Transfer the neural stem cell-like cells to MesenCult~TM medium and continue to culture for 7 - 14 days to obtain differentiated neural cells.

[0073] Biomarkers of exosomal miRNA include hsa-miR-30e-5p.

[0074] The extraction of mesenchymal stem cells specifically includes:

[0075] First, collect mesenchymal stem cells and suspend the mesenchymal stem cells in a medium containing high-concentration sheep serum under sterile conditions;

[0076] Transfer the medium to a low-adhesion culture dish and extract mesenchymal stem cells at 37°C for 24 hours;

[0077] Remove non-adherent cells and residual sheep serum, add serum-free medium, and continue to culture mesenchymal stem cells in a low-adhesion culture dish.

[0078] The components of the modified cell medium include ginsenoside Rg1, notoginseng polysaccharide, vitamin C, EGF, and grape seed extract;

[0079] Among them, the content of ginsenoside Rg1 is 23.08 μg / mL;

[0080] The content of notoginseng polysaccharide is 0.13 μg / ml;

[0081] The content of vitamin C is 3 μg / ml;

[0082] The content of EGF is 0.1 ng / ml;

[0083] The content of grape seed extract is 0.8 μg / ml;

[0084] The content of neuron culture supernatant is 100 μg / mL.

[0085] Mesenchymal stem cells are prepared by extraction from umbilical cord blood.

[0086] The MSC culture medium includes MEM-α basal medium, component A and component B. Among them, component A consists of the following components: exenatide and thrombospondin, and component B consists of human platelet lysate.

[0087] The volume ratio of exenatide in the basal medium is 3%, the volume ratio of thrombospondin in the basal medium is 3.5%, and the volume ratio of platelet lysate in the basal medium is 15%.

[0088] Among them, the same differentiation method as in Example 1 is adopted, and the difference from Example 1 is that the purification of exosomal miRNA is cancelled and the volume ratio of the MSC culture medium is adjusted. Example 3

[0089] The difference from Example 1 is that in S6, when culturing in the improved cell culture medium, an intermittent hypoxic atmosphere culture process is adopted:

[0090] Specifically, it is first cultured in an air atmosphere for 8 h, then cultured in an air atmosphere with 10% CO 2 content for 32 h, then cultured in an air atmosphere with 5% CO 2 content for 24 h, and finally cultured in an air atmosphere for 8 h. Example 4

[0091] The difference from Example 3 is that in S6, when culturing in the improved cell culture medium, a co-culture ultrasound process is adopted:

[0092] Specifically, before culturing, differentiated nerve cells are added in a quantity ratio of 1:1; during culturing, after culturing is completed in an air atmosphere with 10% CO 2 content, it is ultrasonically treated at 200 W for 5 min, and then cultured in an air atmosphere with 5% CO 2 content. Using differentiated nerve cells cultured alone under the same conditions as a control, when calculating the NGF content, the basal NGF (i.e., the NGF produced by differentiated nerve cells) is subtracted.

[0093] Comparative Example 1

[0094] Method for promoting neural differentiation of mesenchymal stem cells by regulating target genes through exosomal miRNA, comprising the following steps:

[0095] S1: Extract mesenchymal stem cells;

[0096] S2: After placing the obtained mesenchymal stem cells into a non-destructive sampling cryopreservation tube, put it into a cryopreservation and recovery box for cryopreservation. The cryopreservation sequence is to cryopreserve in ice water for 1 h, in dry ice for 12 h, and finally transfer to liquid nitrogen for storage;

[0097] S3: After resuscitating the mesenchymal stem cells using the cryopreservation and recovery box, take out the non-destructive sampling cryopreservation tube and detect the cell viability;

[0098] S4: Take the resuscitated mesenchymal cells with cell viability greater than 80% for culture, and culture the mesenchymal stem cells in MSC medium until they cover the culture dish;

[0099] S5: After digesting the mesenchymal stem cells on the culture dish, use Percoll separation solution to separate mononuclear cells;

[0100] S6: Add the separated nuclear cells to the cell culture medium for purification and amplification culture of mesenchymal stem cells for 3 days to obtain neural stem cell-like cells. The cell culture medium is a serum-containing medium;

[0101] S7: Transfer the neural stem cell-like cells to MesenCult~TM medium and continue to culture for 7 - 14 days to obtain differentiated neural cells.

[0102] The biomarker of the exosomal miRNA includes hsa-miR-185-5p.

[0103] Extracting mesenchymal stem cells specifically includes:

[0104] First, collect mesenchymal stem cells and suspend the mesenchymal stem cells in a medium containing high-concentration sheep serum under sterile conditions;

[0105] Transfer the medium to a low-adhesion culture dish and extract mesenchymal stem cells at 37°C for 24 hours;

[0106] Remove non-adherent cells and residual sheep serum, add serum-free medium, and continue to culture mesenchymal stem cells in a low-adhesion culture dish.

[0107] Mesenchymal stem cells are prepared by extracting from one or more of bone marrow, cord blood, cord tissue, placental tissue, and adipose tissue.

[0108] The MSC culture medium comprises MEM-α basal medium, Component A and Component B. Among them, Component A consists of the following components: exenatide and thrombospondin, and Component B consists of human platelet lysate.

[0109] The volume ratio of exenatide in the basal medium is 2%, the volume ratio of thrombospondin in the basal medium is 2.5%, and the volume ratio of platelet lysate in the basal medium is 10%.

[0110] Among them, the differentiation method of Example 2 is adopted, and the difference from Example 1 is that the improved cell culture medium is not used and the volume ratio of the MSC culture medium is adjusted.

[0111] Comparative Example 2

[0112] The difference from Example 1 is that in S6, when culturing in the improved cell culture medium, an intermittent hypoxic atmosphere culture process is adopted:

[0113] Specifically, it is first cultured in an air atmosphere for 8 h, then cultured in an air atmosphere with 5% CO2 content for 32 h, then cultured in an air atmosphere with 10% CO2 content for 24 h, and finally cultured in an air atmosphere for 8 h.

[0114] Comparative Example 3

[0115] The difference from Example 1 is that in S6, when culturing in the improved cell culture medium, an intermittent hypoxic atmosphere culture process is adopted:

[0116] Specifically, it is first cultured in an air atmosphere for 8 h, then cultured in an air atmosphere with 10% CO2 content for 16 h, then cultured in an air atmosphere with 5% CO2 content for 40 h, and finally cultured in an air atmosphere for 8 h.

[0117] Comparative Example 4

[0118] The difference from Example 3 is that in S6, when culturing in the improved cell culture medium, before culturing, differentiated nerve cells in a 1:1 ratio are added. During culturing, after culturing is completed in an air atmosphere with 10% CO2 content, direct culturing in an air atmosphere with 5% CO2 content is carried out. That is, ultrasonic treatment is not performed.

[0119] Select the differentiated nerve cell samples in the above Comparative Example 1, Comparative Example 2, Comparative Example 3, Comparative Example 4, Example 1, Example 2, Example 3, and Example 4, label the antigens in the cells with specific antibodies, then observe under a fluorescence microscope, and detect the cell performance after each sample culture is completed. The results are as follows:

[0120] Example 1 Example 2 Example 3 Example 4 Comparative Example 1 Comparative Example 2 Comparative Example 3 Comparative Example 4 Differentiation efficiency 95% 88% 97% 99% 80% 83% 81% 98% NGF content 100 ng / ml 80 ng / ml 112 ng / ml 127 ng / ml 50 ng / ml 55 ng / ml 53 / ml 119 ng / ml

[0121] As can be seen from the above table, the NGF content, differentiation efficiency, and proliferation and differentiation efficiency of mesenchymal stem cells differentiated by the methods of Example 1 and Example 2 are higher, and the NGF content is 100 ng / ml.

[0122] Among them, compared with Example 1, in Example 2, exosomal miRNAs were not purified, and both its differentiation efficiency and NGF content decreased.

[0123] Among them, compared with Example 2, the differentiation efficiency and NGF content of the comparative example decreased significantly, indicating that when inducing neural differentiation of mesenchymal stem cells, adding exosomal miRNAs and simultaneously improving the culture medium can improve the differentiation efficiency of neural cells and the NGF content.

[0124] The differentiation efficiency and NGF content of Example 3 are significantly better than those of Example 1 and Comparative Examples 2 and 3, indicating the special effect of the intermittent hypoxic atmosphere culture process adopted in Example 3, which requires a high-concentration CO2 atmosphere first and sufficient culture time.

[0125] The differentiation efficiency and NGF content of Example 4 are significantly better than those of Example 3 and Comparative Example 4, indicating the special effect of the ultrasonic co-culture process adopted in Example 4, especially demonstrating the necessity of ultrasonic treatment after a high-concentration CO2 atmosphere.

[0126] In summary, in the present invention, regulating target genes by exosomal miRNAs promotes the neural differentiation of mesenchymal stem cells. By purifying exosomal miRNAs, impurities and interfering substances can be removed. Adding purified exosomal miRNAs during the differentiation of mesenchymal stem cells enables the differentiated neural cells to have high biological activity, which can promote cell proliferation and differentiation, help mesenchymal stem cells grow and differentiate into the desired cell types better, and has an enhanced immunomodulatory effect, can inhibit inflammatory responses, promote tissue repair, and participate in the activation and differentiation processes of immune cells, improving the treatment effect. At the same time, adding purified exosomal miRNAs can maintain biological activity for a long time, and is easy to store and transport, facilitating clinical application.

[0127] It should be noted that in this article, relational terms such as first and second are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the term "comprising", "including" or any other variant thereof is intended to cover non-exclusive inclusion, so that a process, method, article or device comprising a series of elements not only includes those elements, but also includes other elements not expressly listed, or elements inherent to such process, method, article or device.

[0128] Although embodiments of the present invention have been shown and described, it will be understood by those of ordinary skill in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present invention.

Claims

1. A method for promoting the neural differentiation of mesenchymal stem cells by regulating target genes through exosomal miRNAs, characterized in that, The following steps are involved: S1: Extraction of mesenchymal stem cells; S2: After the obtained mesenchymal stem cells are placed in a non-destructive sampling cryopreservation tube, they are placed in a cryopreservation recovery box for cryopreservation, and finally transferred to liquid nitrogen for cryopreservation and storage; S3: After resuscitating the mesenchymal stem cells using a cryopreservation resuscitation box, take out the non-destructive sampling cryopreservation tube and detect the cell activity; S4: Take the revived mesenchymal cells with cell activity greater than 80% for culture, and culture the mesenchymal stem cells with MSC culture medium until they fill the culture dish; S5: After digesting the mesenchymal stem cells on the culture dish, separate the mononuclear cells using Percoll separation solution; S6: Improve the cell culture medium and purify the exosomal miRNA, add the purified exosomal miRNA solution to the improved cell culture medium to purify and expand mesenchymal stem cells, and culture for 3 days to obtain neural stem cell-like cells. When culturing in the improved cell culture medium, use an intermittent low-oxygen atmosphere culture process: specifically, first culture in an air atmosphere for 4-10 h, then culture in an air atmosphere with a 10% CO2 content for 20-40 h, then culture in an air atmosphere with a 5% CO2 content for 15-30 h, and finally culture in an air atmosphere for 4-10 h; S7: Transfer the neural stem cell-like cells to MesenCult medium and continue culturing for 7-14 days to obtain differentiated neural cells; The biomarkers of the exosomal miRNA solution are: hsa-miR-652-3p, hsa-miR-30e-5p or hsa-miR-185-5p; The purification of exosomal miRNA specifically includes: Sample preparation: Transfer the serum sample into a centrifuge tube and take the supernatant after centrifugation; Magnetic beads preparation: Take out the magnetic beads from the kit, wash them and add them to the sample; ‌Incubation‌: Mix the magnetic beads and the sample and incubate; Magnetic bead collection: collect the magnetic beads by centrifugation and magnetic rack, and discard the supernatant; ‌Exosome elution‌: Use buffer to elute the exosomes, and the collected eluate is the purified exosome miRNA solution; The components of the improved cell culture medium include ginsenoside Rg1, notoginseng polysaccharide, vitamin C, EGF and grape seed extract.

2. The method for promoting the neural differentiation of mesenchymal stem cells by regulating target genes through exosomal miRNAs according to claim 1, wherein: The extracting of mesenchymal stem cells specifically comprises: Mesenchymal stem cells are collected and suspended in a culture medium containing high concentration of goat serum under sterile conditions; The culture medium was transferred to a low-adhesion culture dish and the mesenchymal stem cells were extracted at 37°C for 24 h; Non-adherent cells and residual sheep serum were removed, serum-free culture medium was added, and mesenchymal stem cells were cultured in low-adhesion culture dishes.

3. The method for promoting neural differentiation of mesenchymal stem cells by regulating target genes through exosomal miRNAs according to claim 1, characterized in that: The mesenchymal stem cells are obtained by extracting one or more of bone marrow, umbilical cord blood, umbilical cord tissue, placental tissue and adipose tissue.

4. The method for promoting neural differentiation of mesenchymal stem cells by regulating target genes through exosomal miRNAs according to claim 1, characterized in that: The MSC culture medium comprises MEM-α basal culture medium, component A and component B, wherein component A consists of the following components: enalaretide and thrombin, and component B consists of human platelet lysate.

5. The method for promoting neural differentiation of mesenchymal stem cells by regulating target genes through exosomal miRNA according to claim 4, wherein: The volume ratio of exenatide to the basal medium is 0.1% - 5%, the volume ratio of thrombospondin to the basal medium is 0.1% - 5%, and the volume ratio of platelet lysate to the basal medium is 5% - 20%.

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