Method for promoting neural differentiation of mesenchymal stem cells by regulating target gene through exosome miRNA

Abstract

The invention discloses a method for promoting neural differentiation of mesenchymal stem cells by regulating a target gene through exosome miRNA, and belongs to the technical field of biology. The invention discloses a method for promoting neural differentiation of mesenchymal stem cells by regulating a target gene through exosome miRNA. The method comprises the following steps: S1, extracting the mesenchymal stem cells; s2, performing cryopreservation on the obtained mesenchymal stem cells; s3, taking out the lossless sampling cryopreservation tube; S4, culturing by using an MSC culture medium until the culture dish is fully paved with the MSC culture medium; s5, separating mononuclear cells; s6, obtaining neural stem cell-like cells; and S7, obtaining differentiated nerve cells. The invention solves the problem that the application of the mesenchymal stem cells in the treatment of immune-related diseases is influenced because the immune regulation ability of the mesenchymal stem cells cultured in the prior art is reduced, and can promote the proliferation and differentiation of the cells and improve the immune regulation ability of the mesenchymal stem cells.

Description

Technical Field

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

[0002] Mesenchymal stem cells are a type of pluripotent stem cells that have all the common properties of stem cells, namely, self-renewal and multidirectional differentiation capabilities. They are also the most widely used in clinical applications. When used in combination with hematopoietic stem cells, they can improve the success rate of transplantation and accelerate hematopoietic reconstruction. When patients receive high-dose chemotherapy, the infusion of mesenchymal stem cells together with hematopoietic stem cells can significantly accelerate the recovery time of patients' blood cells, and is safe and has no adverse reactions.

[0003] The Chinese patent with publication number CN117363567A discloses a novel method for extracting mesenchymal stem cells, comprising the following steps: step 1: collecting mesenchymal stem cells; step 2: suspending the mesenchymal stem cells in a culture medium containing a high concentration of sheep serum under sterile conditions; step 3: transferring the culture medium to a low-adhesion culture dish; step 4: extracting mesenchymal stem cells at 37°C for 24 hours; step 5: removing non-adherent cells and residual sheep serum, and adding serum-free culture medium; step 6: continuing to culture mesenchymal stem cells in a low-adhesion culture dish; step 7: regularly replacing the serum-free culture medium to maintain the growth and proliferation of mesenchymal stem cells, and ensuring the activity of mesenchymal stem cells by bone marrow, umbilical cord blood, umbilical cord tissue, placental tissue and adipose tissue, so that the survival rate of the culture is 30% higher than that of the traditional culture method, while enhancing the proliferation ability of the mesenchymal stem cells.

[0004] In actual use, the above patents use an important role played by mesenchymal stem cells in tissue repair and regeneration, and this role is partly achieved through the exosomes secreted by them. The mesenchymal stem cells cultured by the above method do not utilize exosomes, which will reduce the immunoregulatory ability of mesenchymal stem cells and affect their application in the treatment of immune-related diseases. This does not meet existing needs. In this regard, we propose a method for promoting the neural differentiation of mesenchymal stem cells by regulating target genes through exosomal miRNA. 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 miRNA, so that the differentiated neural cells have high biological activity, can promote cell proliferation and differentiation, can help mesenchymal stem cells to better grow and differentiate into the desired cell types, and has the effect of improving immunoregulation, can inhibit inflammatory response, promote tissue repair, and participate in the activation and differentiation process of immune cells, thereby improving the therapeutic effect and solving the problems raised in the above-mentioned background technology.

[0006] To achieve the above object, the present invention provides the following technical solution: a method for promoting neural differentiation of mesenchymal stem cells by regulating target genes through exosomal miRNA, comprising the following steps: 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, use Percoll separation solution to separate the mononuclear cells; S6: Improve the cell culture medium and purify the exosomal miRNA, add the purified exosomal miRNA to the improved cell culture medium to purify and expand mesenchymal stem cells for 3 days to obtain neural stem cell-like cells; S7: The neural stem cell-like cells are transferred to MesenCult~TM culture medium and cultured for 7-14 days to obtain differentiated neural cells.

[0007] Preferably, the exosomal miRNA biomarkers include: hsa-miR-652-3p, hsa-miR-30e-5p, hsa-miR-185-5p and hsa-let-7a-5p.

[0008] Preferably, the extracting of mesenchymal stem cells specifically comprises: First, 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; The non-adherent cells and residual goat serum were removed, serum-free medium was added, and the mesenchymal stem cells were continued to be cultured in a low-adhesion culture dish.

[0009] Preferably, the purification of exosomal miRNA specifically includes: Sample preparation: transfer serum and other samples into centrifuge tubes 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.

[0010] Preferably, the components of the improved cell culture medium include ginsenoside Rg1, Panax notoginseng polysaccharide, vitamin C, EGF and grape seed extract.

[0011] Preferably, 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.

[0012] Preferably, the MSC culture medium comprises MEM-α basal medium, component A and component B, wherein component A consists of the following components: enalaretide and thrombospondin, and component B consists of human platelet lysate.

[0013] Preferably, the volume ratio of enalaretide to the basal culture medium is 0.1%-5%, the volume ratio of thrombin spondin to the basal culture medium is 0.1%-5%, and the volume ratio of platelet lysate to the basal culture medium is 5%-20%.

[0014] Compared with the prior art, the present invention has the following beneficial effects: The present invention can remove impurities and interfering substances by purifying exosomal miRNA, and add purified exosomal miRNA when differentiating mesenchymal stem cells, so that 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 has the effect of improving immunomodulation, can inhibit inflammatory response, promote tissue repair, and participate in the activation and differentiation process of immune cells, improve the therapeutic effect, and at the same time, adding purified exosomal miRNA can maintain biological activity for a long time, and is easy to store and transport, which is convenient for clinical application. Among them, the intermittent low oxygen atmosphere culture process and the ultrasonic co-culture process can significantly promote differentiation and increase NGF secretion. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Schematic diagram of the method of the present invention for promoting neural differentiation of mesenchymal stem cells by regulating target genes through exosomal miRNA. DETAILED DESCRIPTION

[0016] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. Example 1

[0017] In order to solve the problem that the existing cultured mesenchymal stem cells do not use 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 , this embodiment provides the following technical solutions: The method for promoting neural differentiation of mesenchymal stem cells by regulating target genes through exosomal miRNA comprises the following steps: 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. The cryopreservation sequence is: cryopreservation in ice water for 2 hours, dry ice for 12 hours, and finally transferred to liquid nitrogen for cryopreservation; Freezing mesenchymal stem cells can not only maintain the vitality and function of mesenchymal stem cells, but also extend the shelf life of mesenchymal stem cells. Living cells transported in culture medium have only a short shelf life, while cryopreservation can significantly extend the storage time of stem cells, allowing them to be used when needed. Cryopreservation can also improve treatment efficiency, and cryopreserved mesenchymal stem cells can still maintain their immune regulation function after thawing. 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, use Percoll separation solution to separate the mononuclear cells; S6: Improve the cell culture medium and purify the exosomal miRNA, add the purified exosomal miRNA to the improved cell culture medium to purify and expand mesenchymal stem cells for 3 days to obtain neural stem cell-like cells; Adding purified exosomal miRNA during the differentiation of mesenchymal stem cells makes the differentiated neural cells highly biologically active, which can promote cell proliferation and differentiation, help mesenchymal stem cells grow and differentiate into the required cell types better, and enhance immunomodulatory effects, inhibit inflammatory responses, promote tissue repair, and participate in the activation and differentiation of immune cells. This immunomodulatory effect helps reduce transplant rejection and improve the safety of cell therapy. As a drug carrier, exosomes have low immunogenicity and good biocompatibility, and can be delivered to target tissues through multiple pathways in the body. As a carrier of drugs or therapeutic factors, it improves the therapeutic effect.

[0018] S7: The neural stem cell-like cells are transferred to MesenCult~TM culture medium and cultured for 7-14 days to obtain differentiated neural cells.

[0019] Exosomal miRNA biomarkers include: hsa-miR-652-3p.

[0020] Extraction of mesenchymal stem cells, including: Mesenchymal stem cells were collected and suspended in a culture medium containing high-concentration goat serum under sterile conditions. The concentration of the goat serum was 10%. 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 medium was added, and mesenchymal stem cells were continued to be cultured in low-adhesion culture dishes. The serum-free medium was replaced regularly to maintain the growth and proliferation of mesenchymal stem cells.

[0021] Purification of exosomal miRNA includes: Sample preparation: transfer serum and other samples into centrifuge tubes 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. The collected eluate is the purified exosome miRNA solution. The buffer is phosphate buffer.

[0022] ‌The benefits of purifying exosomal miRNA mainly include improving the accuracy and reliability of research, as well as discovering new biomarkers and therapeutic targets. ‌By purifying exosomal miRNA, impurities and interfering substances can be removed, making research results more accurate and reliable. In addition, purified exosomal miRNA can also be used for the diagnosis and prognosis assessment of diseases, providing new possibilities for clinical applications.‌ In addition, purified exosomal miRNA can also be used as a potential biomarker for early diagnosis and prognosis assessment of diseases. For example, the serum exosomal miR-223 levels of patients with dementia are correlated with the severity of the disease and can be used as a marker for diagnosis and assessment of disease progression. Similarly, exosomal miRNA has also discovered new biomarkers and treatments in the study of obesity and insulin resistance. These findings provide new possibilities for the treatment and prevention of diseases. ‌ The improved cell culture medium includes ginsenoside Rg1, notoginseng polysaccharide, vitamin C, EGF and grape seed extract, which work together to improve the induction efficiency, and the composition of the culture medium is simple, which is easy to prepare and use; Among them, the content of ginsenoside Rg1 was 23.08 μg / mL; The content of Panax notoginseng polysaccharide was 0.13ug / ml; The content of vitamin C is 3ug / ml; The content of EGF is 0.1ng / ml; The content of grape seed extract is 0.8μg / ml‌.

[0023] Mesenchymal stem cells are extracted from bone marrow.

[0024] 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 spondin, and component B consists of human platelet lysate.

[0025] The volume ratio of enalaretide to the basal culture medium is 5%, the volume ratio of thrombin spondin to the basal culture medium is 5%, and the volume ratio of platelet lysate to the basal culture medium is 20%. Example 2

[0026] The method for promoting neural differentiation of mesenchymal stem cells by regulating target genes through exosomal miRNA comprises the following steps: 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, use Percoll separation solution to separate the mononuclear cells; S6: Improve the cell culture medium, add the exosomal miRNA to the improved cell culture medium to purify and expand the mesenchymal stem cells for 3 days to obtain neural stem cell-like cells; S7: The neural stem cell-like cells are transferred to MesenCult~TM culture medium and cultured for 7-14 days to obtain differentiated neural cells.

[0027] Exosomal miRNA biomarkers include hsa-miR-30e-5p.

[0028] Extraction of mesenchymal stem cells, including: First, 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; Remove non-adherent cells and residual sheep serum, add serum-free medium, and continue to culture mesenchymal stem cells in low-adhesion culture dishes. The ingredients of the improved cell culture medium include ginsenoside Rg1, Panax notoginseng polysaccharide, vitamin C, EGF and grape seed extract; Among them, the content of ginsenoside Rg1 was 23.08 μg / mL; The content of Panax notoginseng polysaccharide was 0.13ug / ml; The content of vitamin C is 3ug / ml; The content of EGF is 0.1ng / ml; The content of grape seed extract is 0.8 μg / ml; The content of neuronal culture supernatant is 100μg / mL. Mesenchymal stem cells are extracted from umbilical cord blood.

[0029] 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 spondin, and component B consists of human platelet lysate.

[0030] The volume ratio of enalaretide to the basal culture medium is 3%, the volume ratio of thrombin-spondin to the basal culture medium is 3.5%, and the volume ratio of platelet lysate to the basal culture medium is 15%. The same differentiation method as in Example 1 was used, except that the purification of exosomal miRNA was eliminated and the volume ratio of the MSC culture medium was adjusted. Example 3

[0031] The difference from Example 1 is that in S6, when culturing in the improved cell culture medium, an intermittent low oxygen atmosphere culturing process is adopted: Specifically, the cells were cultured in an air atmosphere for 8 h and then in a 10% CO 2 The cells were cultured in an atmosphere of 5% CO for 32 h and then in an atmosphere of 5% CO 2 The cells were cultured in an air atmosphere with a low content of 1% CO for 24 h and finally in an air atmosphere for 8 h. Example 4

[0032] The difference from Example 3 is that in S6, when culturing in the improved cell culture medium, a co-culture ultrasonic process is used: Specifically, before culture, differentiated neural cells were added at a ratio of 1:1; during culture, the cells were cultured in an atmosphere of 10% CO 2 After the incubation in an air atmosphere with a content of 5%, the cells were ultrasonically treated at 200W for 5 minutes and then subjected to 5% CO 2 Differentiated neurons cultured alone under the same conditions were used as controls, and the basal NGF (i.e., NGF produced by differentiated neurons) was subtracted when calculating the NGF content.

[0033] Comparative Example 1 The method for promoting neural differentiation of mesenchymal stem cells by regulating target genes through exosomal miRNA comprises the following steps: 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. The cryopreservation sequence is: cryopreservation in ice water for 1 hour, dry ice for 12 hours, and finally transferred to liquid nitrogen for cryopreservation; 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, use Percoll separation solution to separate the mononuclear cells; S6: adding the separated nuclear cells to a cell culture medium to purify and expand the mesenchymal stem cells for 3 days to obtain neural stem cell-like cells, wherein the cell culture medium is a serum-containing culture medium; S7: The neural stem cell-like cells are transferred to MesenCult~TM culture medium and cultured for 7-14 days to obtain differentiated neural cells.

[0034] The exosomal miRNA biomarkers include hsa-miR-185-5p.

[0035] Extraction of mesenchymal stem cells, including: First, 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.

[0036] Mesenchymal stem cells are extracted from one or more of bone marrow, umbilical cord blood, umbilical cord tissue, placental tissue and adipose tissue.

[0037] 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 spondin, and component B consists of human platelet lysate.

[0038] The volume ratio of enalaretide to the basal culture medium is 2%, the volume ratio of thrombin spondin to the basal culture medium is 2.5%, and the volume ratio of platelet lysate to the basal culture medium is 10%. The differentiation method of Example 2 is used, which differs from Example 1 in that the improved cell culture medium is not used and the volume ratio of the MSC culture medium is adjusted.

[0039] Comparative Example 2 The difference from Example 1 is that in S6, when culturing in the improved cell culture medium, an intermittent low oxygen atmosphere culturing process is adopted: Specifically, the cells were cultured in an air atmosphere for 8 h and then in a 5% CO 2 The cells were cultured in an atmosphere of 10% CO for 32 h and then in an atmosphere of 10% CO 2 The cells were cultured in an air atmosphere with a low content of 1% CO for 24 h and finally in an air atmosphere for 8 h.

[0040] Comparative Example 3 The difference from Example 1 is that in S6, when culturing in the improved cell culture medium, an intermittent low oxygen atmosphere culturing process is adopted: Specifically, the cells were cultured in an air atmosphere for 8 h and then in a 10% CO 2 The cells were cultured in an atmosphere of 5% CO for 16 h and then in an atmosphere of 5% CO 2 The cells were cultured in an air atmosphere with a low content of 40 h and finally in an air atmosphere for 8 h.

[0041] Comparative Example 4 The difference from Example 3 is that in S6, when culturing in the improved cell culture medium, differentiated neural cells were added in a 1:1 ratio before culturing. During culturing, the cells were cultured in 10% CO 2 After the incubation in an air atmosphere with 5% CO 2 The cells were cultured in an air atmosphere with a low concentration of 1% sodium bicarbonate. That is, no ultrasonic treatment was performed.

[0042] The differentiated neural cell samples in Comparative Example 1, Comparative Example 2, Comparative Example 3, Comparative Example 4 and Examples 1, 2, 3 and 4 were selected, and the antigens in the cells were labeled with specific antibodies and then observed under a fluorescence microscope. After the culture of each sample was completed, the cell performance was tested, and the results were as follows: 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 100ng / ml 80ng / ml 112ng / ml 127 ng / ml 50ng / ml 55ng / ml 53 / ml 119ng / ml As can be seen from the above table, the NGF content, differentiation efficiency, and proliferation and differentiation efficiency of mesenchymal stem cells differentiated using the methods of Example 1 and Example 2 are higher, and the NGF content is 100 ng / ml.

[0043] Among them, compared with Example 1, Example 2 did not purify the exosomal miRNA, and its differentiation efficiency and NGF content were both reduced.

[0044] Among them, compared with Example 2, the differentiation efficiency and NGF content of the comparative example are greatly reduced, indicating that the addition of exosomal miRNA during the neural differentiation of mesenchymal stem cells and the improvement of the culture medium can improve the neural cell differentiation efficiency and NGF content.

[0045] The differentiation efficiency and NGF content of Example 3 were significantly better than those of Example 1 and Comparative Examples 2 and 3, indicating the special effect of the intermittent low oxygen atmosphere culture process adopted in Example 3, which requires high concentration of CO 2 atmosphere, and at the same time requires sufficient cultivation time.

[0046] The differentiation efficiency and NGF content of Example 4 were significantly better than those of Example 3 and Comparative Example 4, which indicated the special effect of the ultrasonic co-culture process used in Example 4, especially the effect of high concentration of CO 2 Necessity of post-atmosphere ultrasonication.

[0047] In summary, the present invention promotes the neural differentiation of mesenchymal stem cells by regulating target genes through exosomal miRNA. By purifying exosomal miRNA, impurities and interfering substances can be removed. When the mesenchymal stem cells are differentiated, the purified exosomal miRNA is added, so that the differentiated neural cells have high biological activity, which can promote cell proliferation and differentiation, and can help mesenchymal stem cells to better grow and differentiate into the desired cell types. It also has the effect of improving immunoregulation, can inhibit inflammatory response, promote tissue repair, and participate in the activation and differentiation process of immune cells, thereby improving the therapeutic effect. At the same time, the addition of purified exosomal miRNA can maintain biological activity for a long time, and is easy to store and transport, which is convenient for clinical application.

[0048] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device.

[0049] While the embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that many changes, modifications, substitutions and variations can be made to the embodiments without departing from the principles and spirit of the invention.

Claims

1. A method for promoting neural differentiation of mesenchymal stem cells by regulating target genes through exosomal miRNA, 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 to the improved cell culture medium to purify and expand mesenchymal stem cells for 3 days to obtain neural stem cell-like cells; S7: The neural stem cell-like cells are transferred to MesenCult~TM culture medium and cultured for 7-14 days to obtain differentiated neural cells.

2. The method for promoting neural differentiation of mesenchymal stem cells by regulating target genes through exosomal miRNA according to claim 1, characterized in that: The exosomal miRNA biomarkers include: hsa-miR-652-3p, hsa-miR-30e-5p, hsa-miR-185-5p and hsa-let-7a-5p.

3. The method of promoting neural differentiation of mesenchymal stem cells by regulating target genes through exosomal miRNA according to claim 1, characterized in that: 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; The non-adherent cells and residual goat serum were removed, serum-free medium was added, and the mesenchymal stem cells were continued to be cultured in a low-adhesion culture dish.

4. The method of promoting neural differentiation of mesenchymal stem cells by regulating target genes through exosomal miRNA according to claim 1, characterized in that: The purification of exosomal miRNA specifically includes: Sample preparation: transfer serum and other samples into centrifuge tubes 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.

5. The method of promoting neural differentiation of mesenchymal stem cells by regulating target genes through exosomal miRNA according to claim 1, characterized in that: The components of the improved cell culture medium include ginsenoside Rg1, Panax notoginseng polysaccharide, vitamin C, EGF and grape seed extract.

6. The method of claim 1 for promoting neural differentiation of mesenchymal stem cells by regulating target genes through exosomal miRNA, 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.

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

8. The method of promoting neural differentiation of mesenchymal stem cells by regulating target genes through exosomal miRNA according to claim 7, characterized in that: The volume ratio of enalaretide to the basic culture medium is 0.1%-5%, the volume ratio of thrombin spondin to the basic culture medium is 0.1%-5%, and the volume ratio of platelet lysate to the basic culture medium is 5%-20%.

9. The method of claim 1 for promoting neural differentiation of mesenchymal stem cells by regulating target genes through exosomal miRNA, characterized in that: In S6, an intermittent low oxygen atmosphere culture process was used when culturing in the modified cell culture medium: Specifically, the cells were first cultured in an air atmosphere for 4-10 h, then cultured in an air atmosphere with a 10% CO2 content for 20-40 h, then cultured in an air atmosphere with a 5% CO2 content for 15-30 h, and finally cultured in an air atmosphere for 4-10 h.

10. The method of promoting neural differentiation of mesenchymal stem cells by regulating target genes through exosomal miRNA according to claim 9, characterized in that: In S6, a co-culture sonication process was used when culturing in modified cell culture medium: Specifically, before culture, differentiated neural cells were added in a ratio of (0.5-1.5): (0.5-1.5); during culture, after culture in an air atmosphere with a 10% CO2 content, 200W ultrasonic treatment was performed for 3-10 minutes, and then culture was carried out in an air atmosphere with a 5% CO2 content.

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