Preparation method of genetically modified human bone marrow mesenchymal stem cells and application thereof in treatment of nervous system diseases

Abstract

The application belongs to the technical field of biological drugs, and particularly relates to a preparation method of genetically modified human bone marrow mesenchymal stem cells and application thereof in treating nervous system diseases, in particular, multiple sclerosis. The human bone marrow mesenchymal stem cells are double-modified by brain-derived neurotrophic factor (BDNF) and anti-S1PR5 nanobody, so that the activity of the human bone marrow mesenchymal stem cells is improved, the survival of neurons or oligodendrocytes is promoted, cell apoptosis is inhibited, and myelin regeneration is promoted, and the human bone marrow mesenchymal stem cells can be applied to nervous system diseases, in particular, multiple sclerosis.

Description

Technical Field

[0001] This invention relates to human bone marrow mesenchymal stem cells, specifically to a method for preparing genetically modified human bone marrow mesenchymal stem cells and their application in the treatment of nervous system diseases. Background Technology

[0002] In regenerative medicine and clinical treatment, mesenchymal stem cells (MSCs) have become a promising alternative to embryonic stem cells due to their self-renewal and multilineage differentiation capabilities. In recent years, numerous clinical studies have demonstrated the significant therapeutic effects of MSCs on immune diseases, myocardial injury, liver diseases, lung diseases, kidney diseases, and diabetes. Bone marrow mesenchymal stem cells (BMSCs) are an important cell pool involved in tissue regeneration. Under the influence of specific signals induced by tissue damage, BMSCs migrate to the damaged site, aggregate and proliferate locally, and differentiate along different pathways based on different damage signals.

[0003] BMSCs are easy to isolate and expand; however, their proliferative capacity is affected by many factors, including the individual tissue from which they originate, culture conditions, and continuous passage. Unmodified mesenchymal stem cells proliferate slowly in vitro, requiring a long time to expand to clinically applicable scales. Therefore, identifying and cloning genes that regulate the proliferative capacity of mesenchymal stem cells is beneficial not only for their proliferation but also for their widespread clinical application.

[0004] BMSCs are a type of adult stem cell originating from the mesoderm, possessing self-renewal and multi-lineage differentiation potential, capable of differentiating into various cell types such as osteoblasts, chondrocytes, adipocytes, and nerve cells. BMSCs can promote tissue repair and regeneration by secreting various growth factors and cytokines, and have potential applications in the treatment of diseases such as bone defects, nonunion fractures, Parkinson's disease, and spinal cord injuries.

[0005] Brain-derived neurotrophic factor (BDNF) is a crucial neurotrophic factor in the nervous system with multiple important physiological functions: (1) it promotes neuronal growth, differentiation, and survival. By binding to the high-affinity receptor TrkB, it activates signaling pathways such as PI3K / Akt and MAPK / ERK, enhancing the anti-apoptotic ability of neurons; (2) it has significant neuroprotective functions, helping neurons resist various injuries, such as cerebral ischemia and neurotoxins. In addition, BDNF also promotes neuronal migration and synaptic remodeling by regulating the dynamic changes of the cytoskeleton; (3) it can promote the reorganization of neural networks and help restore function. For example, in a chronic cerebral ischemia model, increased BDNF expression helps alleviate neurodegeneration.

[0006] S1PR5 (sphingosine 1-phosphate receptor 5) is a G protein-coupled receptor belonging to the sphingosine 1-phosphate receptor (S1PR) family and is involved in various physiological processes. Studies have shown that S1PR5 primarily functions by regulating lymphocyte migration and distribution. In multiple sclerosis (MS), S1PR5 activation can prevent lymphocytes from efflux from lymph nodes, thereby reducing the entry of inflammatory cells into the central nervous system and alleviating the inflammatory response. Furthermore, it may also play a protective role against neurodegenerative changes by crossing the blood-brain barrier and promoting myelin regeneration and nerve repair. Currently, selective S1P1 and S1P5 receptor modulators have been approved for the treatment of relapsing-remitting MS (including clinically isolated syndrome, relapsing-remitting MS, and relapsed secondary progressive MS).

[0007] Currently, there are no reported studies on the combined use of BDNF and S1PR5-targeting nanobodies to modify human bone marrow mesenchymal stem cells for the treatment of multiple sclerosis. Therefore, further research is needed to explore effective treatments for multiple sclerosis. Summary of the Invention

[0008] Based on the shortcomings of existing technologies, the purpose of this invention is to combine BDNF protein and nanobodies targeting S1PR5, and modify them into human bone marrow mesenchymal stem cells to treat multiple sclerosis.

[0009] One aspect of the present invention is to provide a human bone marrow mesenchymal stem cell, specifically by modifying human bone marrow mesenchymal stem cells with BDNF protein and nanobodies targeting S1PR5.

[0010] Preferably, the amino acid sequence of BDNF is shown in SEQ ID No. 1, and its nucleotide sequence is shown in SEQ ID No. 3.

[0011] Preferably, the amino acid sequence of SEQ ID No. 1 is as follows:

[0012] HSDPARRGELSVCDSISEWVTAADKKTAVDMSGGTVTVLEKVPVSKGQLKQYFYETKCNPMGYTKEGCRGIDKRHWNSQCRTTQSYVRALTMDSKKRIGWRFIRIDTSCVCTLTIKRGR.

[0013] Preferably, the nucleotide sequence of SEQ ID No. 3 is as follows:

[0014] catagcgatccggcgcgccgcggcgaactgagcgtgtgcgatagcattagcgaatgggtg

[0015] accgcggcggataaaaaaaccgcggtggatatgagcggcggcaccgtgaccgtgctggaa

[0016] aaagtgccggtgagcaaaggccagctgaaacagtatttttatgaaaccaaatgcaacccg

[0017] atgggctatacccaaagaaggctgccgcggcattgataaacgccattggaacagccagtgc

[0018] cgcaccacccagagctatgtgcgcgcgctgaccatggatagcaaaaaacgcattggctgg

[0019] cgctttattcgcattgataccagctgcgtgtgcaccctgaccattaaacgcggccgc.

[0020] Preferably, the amino acid sequence of the anti-S1PR5 nanobody is shown in SEQ ID No. 2, and its nucleotide sequence is shown in SEQ ID No. 4.

[0021] Preferably, the amino acid sequence of SEQ ID No. 2 is as follows:

[0022] EVQLVQSGAEVKKPGESLKISCQSFGYIFITIWFHWMRQMPGQGLEWMGFQSSTLVSIRGERIPHQVTISADKSSSTAYLQWSSLKASDTAMYFCARFFGHIRETHGVQWGQGTMVTVSS.

[0023] Preferably, the nucleotide sequence of SEQ ID No. 4 is as follows:

[0024] gaagtgcagctggtgcagagcggcgcggaagtgaaaaaaccgggcgaaagcctgaaaatt

[0025] agctgccagagctttggctatatttttattaccatttggtttcattggatgcgccagatg

[0026] ccgggccagggcctggaatggatgggctttcagagcagcaccctggtgagcattcgcggc

[0027] gaacgcattccgcatcaggtgaccattagcgcggataaaagcagcagcaccgcgtatctg

[0028] cagtggagcagcctgaaagcgagcgataccgcgatgtatttttgcgcgcgcttttttggc

[0029] catattcgcgaaacccatggcgtgcagtggggccagggcaccatggtgaccgtgagcagc.

[0030] Preferably, the BDNF gene is obtained by optimization based on GENBANK number BAO51936.1.

[0031] Preferably, the specific steps of the preparation method of the anti-S1PR5 nanobody are as follows:

[0032] (1) Immunization schedule

[0033] Alpacas were immunized with the full-length S1PR5 protein (GenBank: AAH67781.1) as the antigen. The total antigen dose for each immunization was maintained between 1-2 mg and the volume was less than 2 mL. Before immunization, the antigen and adjuvant were emulsified 1:1 to form a homogeneous mixture and stored at 4°C. Then, the ear tags of blank alpacas were recorded to begin the immunization experiment. Each time, injections were made on both sides near the lymph nodes in the alpaca's neck, with two injection points on each side. Approximately 0.4 mL of emulsified antigen was injected at each point. After immunization, the alpacas were observed for half an hour to confirm that they were in good condition and had no adverse symptoms. Immunization was performed every 2 weeks, for a minimum of 4 immunizations.

[0034] (2) Antibody gene acquisition

[0035] Blood (50 mL) was collected from the jugular vein of the alpaca 5-7 days after the fourth immunization. Lymphocytes were isolated, RNA was extracted and reverse transcribed into cDNA. Primers were designed based on the conserved region of the heavy chain antibody, and the variable region gene of the heavy chain antibody was amplified by PCR. Upstream primer: 5'cggcgcggaagtgaaaaa3', downstream primer: 5'gccatgggtttcgcgaatat3'. The PCR system consisted of 2 μL cDNA, 2 μL each of upstream and downstream primers, 0.25 μL Taq DNA Polymerase Hot Start enzyme, 5 μL 10X Taq Buffer, 4 μL dNTPs, and sterile water to a final volume of 50 μL. The reaction program was as follows: pre-denaturation: 98℃ for 3 minutes; cyclic amplification: 95℃ for 30 seconds, 57℃ for 30 seconds, 68℃ for 40 seconds, increasing by 2 seconds per cycle, repeated for 22 cycles; annealing extension: 68℃ for 5 minutes. Finally, the bands were observed by agarose gel electrophoresis.

[0036] (3) Phage library construction and screening

[0037] The correctly amplified antibody gene fragment was ligated to a phage particle to construct a phage display library. Then, using phage display screening technology, based on the principle of specific binding between the antigen and the antibody displayed on the phage surface, multiple rounds of screening were conducted to select phage clones that could specifically bind to the target antigen from the library, thereby obtaining the corresponding nanobody gene, the nucleotide sequence of which is shown in SEQ ID No. 4.

[0038] (4) Expression and purification

[0039] The selected nanobody genes are cloned into a suitable expression vector (plasmid vector) and then transformed into host cells for expression. The expressed nanobodies can be purified by affinity chromatography.

[0040] Another aspect of the present invention is to provide a method for preparing human bone marrow mesenchymal stem cells, the specific steps of which include:

[0041] S1. Preparation of human bone marrow mesenchymal stem cells: Bone marrow tissue containing primary mesenchymal stem cells was taken, cut into small pieces, and washed with phosphate buffer on a culture dish until the washing solution became clear. The pieces were then thoroughly minced. The minced tissue was transferred to a 50 ml centrifuge tube, and 25 ml of collagenase IV (0.1% W / v) was added. The tube was then incubated at 37°C with shaking for 30 min. 20 ml of phosphate buffer was added to the digested tissue, mixed well, and filtered through a 100 μm sieve. The filtrate was centrifuged at 1200 rpm for 5 min at 4°C. The supernatant was discarded, and the cell pellet was retained. 5 ml of DMEM medium containing 20% ​​fetal bovine serum was added to the cell pellet, mixed well, and then seeded into cell culture flasks. The cells were cultured at 37°C in 5% CO2, with the medium changed every 2 days for passage.

[0042] S2. Construction of lentiviral vectors: Based on the nucleotide sequences of SEQ ID No. 3 and SEQ ID No. 4, BDNF and anti-S1PR5 nanobody nucleotide sequences were directly synthesized, ligated into the enzyme-digested lentiviral expression vector pEF1α, and recombinant plasmids were constructed. These plasmids were then transformed into DH5α competent cells for amplification. The plasmids were extracted and sequenced for verification, and the successfully constructed plasmids were preserved. 293T cells in the logarithmic growth phase were digested to prepare a cell suspension and seeded into 24-well culture plates. When the cell confluence reached approximately 70-80%, 293T cells were co-transfected with plasmid DNA, the packaging plasmid psPAX2 vector, the envelope plasmid pMD2G vector, and LIPOFECTAMINE 3000. After 6 hours of culture, the medium was replaced with fresh medium, and the cells were cultured for another 72 hours. The lentivir-rich 293T cell supernatant was collected, filtered, centrifuged, and concentrated to obtain a high-titer viral concentrate.

[0043] S3. Take BMSCs in the logarithmic growth phase (passage 5 or less) and seed them at 100,000 cells / well in a 12-well culture plate with 1 ml of culture medium per well. When the cell confluence is approximately 50%, remove the culture medium, wash with PBS, and transfect with the virus concentrate from step S2 for 10 hours. After transfection, change the culture medium and continue culturing and passage. Select BMSCs stably expressing BDNF and anti-S1PR5 nanobodies using puromycin-containing medium for 7 days, changing the medium every 2 days.

[0044] Another aspect of the present invention is to provide an application of human bone marrow mesenchymal stem cells, specifically for the treatment of neurological diseases, particularly multiple sclerosis. Modifying human bone marrow mesenchymal stem cells with BDNF and anti-S1PR5 nanobodies can effectively treat multiple sclerosis.

[0045] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0046] This invention is the first to modify human bone marrow mesenchymal stem cells with BDNF and anti-S1PR5 nanobodies. BDNF can promote the proliferation and activity of human bone marrow mesenchymal stem cells, while anti-S1PR5 nanobodies bind to S1PR5, promoting the survival of neurons or oligodendrocytes, inhibiting apoptosis, promoting myelin regeneration, and improving the therapeutic effect of multiple sclerosis. Attached Figure Description

[0047] Figure 1 Preparation process of conventional nanobodies

[0048] Figure 2 Western blot diagram of β3-tubulin and Oligo2 protein expression Detailed Implementation

[0049] The present invention will be further described below with reference to specific implementation examples, but the present invention is not limited to these embodiments.

[0050] Example 1: Preparation of anti-S1PR5 nanobodies

[0051] (1) Immunization schedule

[0052] Alpacas were immunized with the full-length S1PR5 protein (GenBank: AAH67781.1) as the antigen. The total antigen dose for each immunization was maintained between 1-2 mg and the volume was less than 2 mL. Before immunization, the antigen and adjuvant were emulsified 1:1 to form a homogeneous mixture and stored at 4°C. Then, the ear tags of blank alpacas were recorded to begin the immunization experiment. Each time, injections were made on both sides near the lymph nodes in the alpaca's neck, with two injection points on each side. Approximately 0.4 mL of emulsified antigen was injected at each point. After immunization, the alpacas were observed for half an hour to confirm that they were in good condition and had no adverse symptoms. Immunization was performed every 2 weeks, for a minimum of 4 immunizations.

[0053] (2) Antibody gene acquisition

[0054] Blood (50 mL) was collected from the jugular vein of the alpaca 5-7 days after the fourth immunization. Lymphocytes were isolated, RNA was extracted and reverse transcribed into cDNA. Primers were designed based on the conserved region of the heavy chain antibody, and the variable region gene of the heavy chain antibody was amplified by PCR. Upstream primer: 5'cggcgcggaagtgaaaaa3', downstream primer: 5'gccatgggtttcgcgaatat3'. The PCR system consisted of 2 μL cDNA, 2 μL each of upstream and downstream primers, 0.25 μL Taq DNA Polymerase Hot Start enzyme, 5 μL 10X Taq Buffer, 4 μL dNTPs, and sterile water to a final volume of 50 μL. The reaction program was as follows: pre-denaturation: 98℃ for 3 minutes; cyclic amplification: 95℃ for 30 seconds, 57℃ for 30 seconds, 68℃ for 40 seconds, increasing by 2 seconds per cycle, repeated for 22 cycles; annealing extension: 68℃ for 5 minutes. Finally, the bands were observed by agarose gel electrophoresis.

[0055] (3) Construction and screening of phage libraries

[0056] The correctly amplified antibody gene fragment was ligated to a phage particle to construct a phage display library. Then, using phage display screening technology, based on the principle of specific binding between the antigen and the antibody displayed on the phage surface, multiple rounds of screening were conducted to select phage clones that could specifically bind to the target antigen from the library, thereby obtaining the corresponding nanobody gene, the nucleotide sequence of which is shown in SEQ ID No. 4.

[0057] (4) Expression and purification

[0058] The selected nanobody genes are cloned into a suitable expression vector (plasmid vector) and then transformed into host cells for expression. The expressed nanobodies can be purified by affinity chromatography.

[0059] Example 2: Preparation of mesenchymal stem cells modified with BDNF and anti-S1PR5 nanobodies

[0060] S1. Preparation of primitive human bone marrow mesenchymal stem cells: Bone marrow tissue containing primary mesenchymal stem cells was taken, cut into small pieces, and washed with phosphate buffer on a culture dish until the washing solution became clear. The pieces were then thoroughly minced. The minced tissue was transferred to a 50 ml centrifuge tube, and 25 ml of collagenase IV (0.1% W / v) was added. The tube was then incubated at 37°C with shaking for 30 min. 20 ml of phosphate buffer was added to the digested tissue, mixed well, and filtered through a 100 μm sieve. The filtrate was centrifuged at 1200 rpm for 5 min at 4°C. The supernatant was discarded, and the cell pellet was retained. 5 ml of DMEM medium containing 20% ​​fetal bovine serum was added to the cell pellet, mixed well, and then seeded into cell culture flasks. The cells were cultured at 37°C in 5% CO2, with the medium changed every 2 days for passage.

[0061] S2. Construction of Lentiviral Vectors: Based on the nucleotide sequences of SEQ ID No. 3 and SEQ ID No. 4, BDNF and anti-S1PR5 nanobody nucleotide sequences were directly synthesized and ligated into the enzyme-digested lentiviral expression vector pEF1α to construct recombinant plasmids. These plasmids were then transformed into DH5α competent cells for amplification. The plasmids were extracted and sequenced for verification, and the successfully constructed plasmids were preserved. 293T cells in the logarithmic growth phase were digested to prepare a cell suspension and seeded into 24-well culture plates. When the cell confluence reached approximately 70-80%, 293T cells were co-transfected with plasmid DNA, the packaging plasmid psPAX2 vector, the envelope plasmid pMD2G vector, and LIPOFECTAMINE 3000. After 6 hours of culture, the medium was replaced with fresh medium, and the cells were cultured for another 72 hours. The lentivirus-rich 293T cell supernatant was collected, filtered, centrifuged, and concentrated to obtain a high-titer virus concentrate.

[0062] S3. Preparation of Genetically Modified Mesenchymal Stem Cells: BMSCs in the logarithmic growth phase (passage 5 or less) were seeded at 100,000 cells / well in 12-well culture plates, with 1 ml of culture medium per well. When cell confluence was approximately 50%, the culture medium was removed, and the cells were washed with PBS and transfected with the concentrated virus solution from step S2 for 10 hours. The culture medium was then replaced, and the cells were passaged using standard methods. Selection was performed using puromycin-containing medium for 7 days, with medium changes every 2 days, to obtain BMSCs stably expressing BDNF and anti-S1PR5 nanobodies.

[0063] Comparative Example 1: Preparation of BDNF-modified mesenchymal stem cells

[0064] The difference from Example 1 is that when constructing the lentiviral vector, only the BDNF gene is introduced into the plasmid to construct the recombinant plasmid; the other steps are the same.

[0065] Comparative Example 2: Preparation of mesenchymal stem cells modified with anti-S1PR5 nanobodies

[0066] The difference from Example 1 is that when constructing the lentiviral vector, only the gene for the anti-S1PR5 nanobody is introduced into the plasmid to construct the recombinant plasmid; the remaining steps are the same.

[0067] Example 3: Application of human bone marrow mesenchymal stem cells in the treatment of multiple sclerosis

[0068] 1. Construction of an animal model of multiple sclerosis

[0069] An experimental autoimmune encephalomyelitis (EAE) model, similar in pathological features to multiple sclerosis, was used for modeling. The specific steps are as follows:

[0070] SPF-grade female C57BL / 6 mice aged 10-14 weeks were selected and divided into four groups of six mice each. These groups were: Group A (model control group), Group B (model + BDNF-modified human bone marrow mesenchymal stem cell therapy group), Group C (model + anti-S1PR5 nanobody-modified human bone marrow mesenchymal stem cell therapy group), and Group D (model + BDNF and anti-S1PR5 nanobody dual-modified human bone marrow mesenchymal stem cell therapy group). MOG (myelin oligodendrocyte glycoprotein) 35-55 peptide was used as an antigen, mixed with complete Freund's adjuvant, and injected subcutaneously into the peritoneum at a dose of 200 μg per mouse. Motor incoordination, limb paralysis, and sensory abnormalities appeared 7-14 days after injection, confirming successful modeling.

[0071] 2. Collect total protein and behavioral scores after drug treatment.

[0072] Treatment was initiated 14 days after the onset of symptoms. The administration regimen was as follows: 1 mL of BMSCs (10...) was administered once daily to each mouse via intraperitoneal injection. 5 (cells / mL), administration continued for 14 days.

[0073] Fourteen days after administration, spinal cord samples were taken from three birds in each group for protein extraction. Western blot analysis was performed on neuronal marker protein β3-tubulin and oligodendrocyte marker protein Olig2.

[0074] Fourteen days after administration, three animals from each group were selected for behavioral scoring to verify the treatment effect.

[0075] 3. Effectiveness Evaluation

[0076] Western blot analysis showed that the protein expression of β3-tubulin and Olig2 in group D was higher than that in other groups; group B, due to modification with brain-derived neurotrophic factor, showed higher β3-tubulin protein expression compared to groups A and C; and group C, due to modification with anti-S1PR5 nanobody, showed higher Olig2 protein expression compared to groups A and B.

[0077] According to the behavioral scores (Table 1), Group D had better health and behavioral activity than the other groups, Group A had the worst health and behavioral activity, while Groups B and C showed some limitations in their behavioral activity.

[0078] Table 1

[0079]

[0080] Notes: 0 points: Asymptomatic; 1 point: Tail weakness or paralysis; 2 points: Hind limb weakness, but able to walk normally; 3 points: Hind limb paralysis, unable to walk; 4 points: Quadriplegia, unable to move voluntarily; 5 points: Death.

[0081] in conclusion

[0082] BMSCs modified with BDNF and anti-S1PR5 nanobodies not only enhance the activity of human bone marrow mesenchymal stem cells in the treatment of multiple sclerosis, but also promote the survival of neurons or oligodendrocytes, inhibit apoptosis, and promote myelin regeneration, thus effectively treating multiple sclerosis.

[0083] The basic principles, main features, and advantages of this invention have been described above. Those skilled in the art should understand that this invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely principles of the invention. Various changes and modifications can be made without departing from the spirit and scope of the invention, and all such changes and modifications fall within the scope of the invention as claimed. The scope of protection claimed by this invention is defined by the appended claims and their equivalents.

Claims

1. A genetically modified human mesenchymal stem cell, wherein, The human bone marrow mesenchymal stem cells are obtained by double modification of brain-derived neurotrophic factor (BDNF) and anti-S1PR5 nanobody, wherein the amino acid sequence of the BDNF is shown as SEQ ID No. 1, and the amino acid sequence of the anti-S1PR5 nanobody is shown as SEQ ID No. 2; the human bone marrow mesenchymal stem cells are obtained by the following method: taking bone marrow tissue containing primary mesenchymal stem cells, cutting small pieces, washing with phosphate buffer on a culture dish until the washing liquid is transparent, and then cutting thoroughly; transferring the cut tissue to a 50ml centrifuge tube, adding 25ml of 0.1% mass / volume concentration of collagenase IV, and placing in a 37℃ constant temperature box for shaking digestion for 30min; adding 20ml of phosphate buffer to the digested tissue, mixing, and then filtering through a 100um sieve, centrifuging the filtrate at 1200rpm / min in a 4℃ centrifuge for 5min, removing the supernatant and retaining the cell pellet; adding 5ml of 20% fetal bovine serum DMEM medium to the cell pellet, mixing, and then inoculating into a cell bottle, and culturing at 37℃, 5% CO2, replacing the liquid every 2 days, and subculturing.

2. The human mesenchymal stem cell of claim 1, wherein, The BDNF protein is obtained by optimization based on GENBANK No. BAO51936.

1.

3. The human bone marrow mesenchymal stem cell of claim 1, wherein, The preparation method of the anti-S1PR5 nanobody is: (1) Immunization program The full-length protein GenBank: AAH67781.1 of S1PR5 is used as an antigen to immunize a llama, the total antigen amount is kept between 1-2mg per immunization, the volume is below 2mL, the antigen and adjuvant are emulsified 1:1 before immunization to form a uniform mixture, which is stored at 4℃; then the ear number of the blank llama is recorded to start the immunization experiment, each time the llama is injected in the left and right sides of the neck lymph nodes, 2 points per side, 0.4mL emulsified antigen per point, the llama is observed for half an hour after immunization to confirm that the llama is in good condition without discomfort, the immunization is performed every 2 weeks, and at least 4 times of immunization are performed; (2) Antibody gene acquisition Blood 50mL is collected from the jugular vein of the llama 5-7 days after the fourth immunization, the lymphocytes are separated, the RNA is extracted and reverse transcribed into cDNA, the primers are designed according to the conserved region of the heavy chain antibody, and the variable region gene of the heavy chain antibody is obtained by PCR amplification; the upstream primer is 5'cggcgcggaagtgaaaaa3', and the downstream primer is 5'gccatgggtttcgcgaatat3'; the PCR system is cDNA 2ul, the upstream and downstream primers are each 2ul, Taq DNA Polymerase Hot Start enzyme 0.25ul, 10X Taq Buffer 5ul, dNTP 4ul, and sterile water is supplemented to 50ul; the reaction program is pre-denaturation: 98℃ for 3min; cycle amplification: 95℃ for 30s, 57℃ for 30s, 68℃ for 40s, each cycle increases by 2s, repeats for 22 cycles; annealing extension: 68℃ for 5min; finally, the bands are observed by agarose gel electrophoresis; (3) Phage library construction and screening The antibody gene fragment correctly amplified is connected to a phagemid to construct a phage display library; then through phage display screening technology, using the principle that antigens specifically bind to antibodies displayed on the surface of phages, after multiple rounds of screening, phage clones capable of specifically binding to target antigens are screened from the library, so that the corresponding nanobody gene is obtained, and the nucleotide sequence of the nanobody gene is shown as SEQ ID No. 4; (4) Expression and purification The nanobody gene screened is cloned into a suitable expression vector, and then transformed into host cells for expression; the nanobody after expression is purified by affinity chromatography.

4. A method of preparing the genetically modified human mesenchymal stem cell of claim 1, comprising the steps of, The method comprises the following steps: S1, preparing human bone marrow mesenchymal stem cells: taking bone marrow tissue containing primary mesenchymal stem cells, cutting small pieces, washing with phosphate buffer until the washing liquid is transparent on a culture dish, and then cutting the pieces thoroughly; the cut tissue is transferred to a 50ml centrifuge tube, 25ml of 0.1% collagenase IV is added, and it is shaken in a 37℃ constant temperature box for 30min; 20ml of phosphate buffer is added to the digested tissue, mixed and filtered through a 100μm sieve, the filtrate is centrifuged at 1200r / min for 5min in a 4℃ centrifuge, the supernatant is removed and the cell pellet is retained; 5ml of 20% fetal bovine serum DMEM medium is added to the cell pellet, mixed and inoculated into a cell bottle, cultured at 37℃ and 5% CO2, and the medium is changed every 2 days for subculture; S2, constructing a lentivirus vector; synthesizing BDNF and anti-S1PR5 nanobody nucleotide sequences according to the nucleotide sequences of SEQ ID No. 3 and SEQ ID No. 4, connecting them into the enzyme-digested lentivirus expression vector pEF1α, constructing a recombinant plasmid, transforming DH5α competent cells for amplification; extracting the plasmid for sequencing verification, and storing the successfully constructed plasmid; logarithmic phase 293T cells are digested to prepare a cell suspension and inoculated on a 24-well culture plate, cultured in a cell incubator until the cell confluence rate is 70-80%, and then the plasmid DNA, packaging plasmid psPAX2 vector, envelope plasmid pMD2G vector and LIPOFECTAMINE 3000 are co-transfected into 293T cells, the culture medium is replaced after 6h of culture, and the 293T cell supernatant rich in lentivirus is collected after 72h of continuous culture, filtered, centrifuged and concentrated to obtain a high-titer virus concentrate; S3, taking BMSCs in the logarithmic growth phase within 5 generations, inoculating 100,000 cells / well in a 12-well culture plate, and adding 1ml of culture medium per well; when the cell confluence is 50%, the culture medium is removed, washed with PBS, and then transfected with the virus concentrate of step S2 for 10h; the culture medium is replaced after transfection and the cells are cultured and subcultured; the culture medium containing puromycin is used for screening for 7d, and the medium is changed every 2d to obtain BMSCs stably expressing BDNF and anti-S1PR5 nanobodies.

5. The preparation method according to claim 4, characterized in that, The sequence encoding BDNF is shown as SEQ ID No. 3, and the sequence encoding the anti-S1PR5 nanobody is shown as SEQ ID No.

4.

6. The use of the human mesenchymal stem cell of claim 1 in the preparation of a medicament for treating a nervous system disease, characterized in that, The neurological disease is multiple sclerosis.

7. Use according to claim 6, characterized in that, The human bone marrow mesenchymal stem cells promote survival of neurons and oligodendrocytes, inhibit apoptosis, and promote remyelination.

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