CN120173888B

CN120173888B - A method for preparing genetically modified human bone marrow mesenchymal stem cells and its application in the treatment of nervous system diseases.

Info

Publication number: CN120173888B
Application number: CN202510363242.5A
Authority: CN
Inventors: 王翔; 韦燕; 何咏欣
Original assignee: Guangzhou Tongshi Medical Research Co ltd
Current assignee: Guangzhou Tongshi Medical Research Co ltd
Priority date: 2025-03-26
Filing date: 2025-03-26
Publication date: 2025-12-30
Anticipated expiration: 2045-03-26
Also published as: CN120173888A

Abstract

The invention belongs to the technical field of biological medicines, and in particular relates to a preparation method of a genetically modified human bone marrow mesenchymal stem cell and application thereof in the aspect of treating nervous system diseases, in particular to multiple sclerosis. The invention can improve the activity of human marrow mesenchymal stem cells, promote the survival of neurons or oligodendrocytes, inhibit apoptosis and promote the regeneration of myelin sheath by the double modification of brain-derived neurotrophic factor (BDNF) and anti-S1 PR5 nanometer antibody, and can be applied to nervous system diseases, in particular to multiple sclerosis.

Description

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

Technical Field

The invention relates to a human bone marrow mesenchymal stem cell, in particular to a preparation method of a genetically modified human bone marrow mesenchymal stem cell and application thereof in treating nervous system diseases.

Background

In regenerative medicine and clinical treatment, because the mesenchymal stem cells have the capability of self-renewal and multi-lineage differentiation, the mesenchymal stem cells become promising substitutes for embryonic stem cells, and in recent years, clinical researches have been carried out successively to prove that the mesenchymal stem cells have obvious therapeutic effects on immune diseases, myocardial injury, liver diseases, lung diseases, kidney diseases and diabetes. Bone mesenchymal stem cells (Bone Marrow MESENCHYMAL STEM CELLS, BMSCS) are an important cell bank involved in tissue regeneration. Under the action of special signals caused by tissue injury, BMSCs migrate to the injured part, locally accumulate and proliferate, and differentiate along different paths according to different injury signals.

BMSCs are susceptible to isolated expansion, however their proliferative capacity is affected by a number of factors including tissue derived from individuals, culture conditions and sustained passage. The mesenchymal stem cells without genetic modification have slower proliferation rate in vitro and longer time for amplifying to clinical application orders of magnitude. Therefore, the genes related to the proliferation capacity of the mesenchymal stem cells are searched and cloned for expression regulation, which is beneficial to the proliferation of the mesenchymal stem cells and the wide application of clinical treatment.

BMSCs are a class of adult stem cells derived from the mesoderm that have self-renewing and multipotent differentiation potential and are capable of differentiating into a variety of cell types, such as osteoblasts, chondrocytes, adipocytes, neural cells, and the like. BMSCs can promote tissue repair and regeneration by secreting various growth factors, cytokines and the like, and are widely applied to the treatment of bone defects, fracture nonunion, parkinsonism, spinal cord injury and other diseases and have potential application value.

Brain-derived neurotrophic factor (Brain-Derived Neurotrophic Factor, BDNF), a neurotrophic factor that is critical in the nervous system, has a variety of important physiological functions, and (1) is capable of promoting the growth, differentiation and survival of neurons. The anti-apoptosis ability of neurons is enhanced by combining with a high affinity receptor TrkB, activating signal paths such as PI3K/Akt, MAPK/ERK and the like, and (2) the anti-apoptosis ability of neurons is enhanced, and the anti-apoptosis agent has an obvious neuroprotection function and can help the neurons resist various injuries such as cerebral ischemia, neurotoxin and the like. In addition, BDNF can promote migration and synaptic reconstruction of neurons by regulating dynamic change of cytoskeleton, and (3) can promote reorganization of neural network and help recovery of functions. For example, in a chronic brain ischemia model, increased expression of BDNF helps to reduce neurodegeneration.

S1PR5 (sphingosine-1-phosphate receptor 5) is a G protein coupled receptor, belongs to the family of sphingosine-1-phosphate receptors (S1 PR), and is involved in a variety of physiological processes. Studies have shown that S1PR5 acts primarily by regulating lymphocyte migration and distribution. In multiple sclerosis, activation of S1PR5 can prevent lymphocyte excretion from the lymph node, thereby reducing inflammatory cell entry into the central nervous system and alleviating inflammatory responses. In addition, it is also possible to protect neurodegenerative changes by promoting remyelination and nerve repair through the blood brain barrier. Currently, selective S1P1 and S1P5 receptor modulators have been approved for the treatment of relapsing multiple sclerosis (including clinically isolated syndrome, relapsing-remitting MS, and relapsing-secondary progressive MS).

At present, no study on the combined modification of human bone marrow mesenchymal stem cells by combining BDNF and a nanobody for S1PR5 for the treatment of multiple sclerosis has been reported. Therefore, there is a need for effective treatment of multiple sclerosis for this development.

Disclosure of Invention

Based on the shortcomings in the prior art, the invention aims to combine BDNF protein and a nano antibody aiming at S1PR5, modify the BDNF protein and the nano antibody to human bone marrow mesenchymal stem cells and treat multiple sclerosis.

One aspect of the present invention provides a human bone marrow mesenchymal stem cell, in particular, a BDNF protein and a nanobody for S1PR5 are modified on the human bone marrow mesenchymal stem cell.

Preferably, the BDNF has an amino acid sequence shown as SEQ ID No.1 and a nucleotide sequence shown as SEQ ID No. 3.

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

HSDPARRGELSVCDSISEWVTAADKKTAVDMSGGTVTVLEKVPVSKGQLKQYFYETKCNPMGYTKEGCRGIDKRHWNSQCRTTQSYVRALTMDSKKRIGWRFIRIDTSCVCTLTIKRGR.

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

catagcgatccggcgcgccgcggcgaactgagcgtgtgcgatagcattagcgaatgggtg

accgcggcggataaaaaaaccgcggtggatatgagcggcggcaccgtgaccgtgctggaa

aaagtgccggtgagcaaaggccagctgaaacagtatttttatgaaaccaaatgcaacccg

atgggctataccaaagaaggctgccgcggcattgataaacgccattggaacagccagtgc

cgcaccacccagagctatgtgcgcgcgctgaccatggatagcaaaaaacgcattggctgg

cgctttattcgcattgataccagctgcgtgtgcaccctgaccattaaacgcggccgc。

preferably, the amino acid sequence of the anti-S1 PR5 nano antibody is shown as SEQ ID No.2, and the nucleotide sequence of the anti-S1 PR5 nano antibody is shown as SEQ ID No. 4.

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

EVQLVQSGAEVKKPGESLKISCQSFGYIFITIWFHWMRQMPGQGLEWMGFQSSTLVSIRGERIPHQVTISADKSSSTAYLQWSSLKASDTAMYFCARFFGHIRETHGVQWGQGTMVTVSS.

preferably, the nucleotide sequence of SEQ ID No.4 is as follows:

gaagtgcagctggtgcagagcggcgcggaagtgaaaaaaccgggcgaaagcctgaaaatt

agctgccagagctttggctatatttttattaccatttggtttcattggatgcgccagatg

ccgggccagggcctggaatggatgggctttcagagcagcaccctggtgagcattcgcggc

gaacgcattccgcatcaggtgaccattagcgcggataaaagcagcagcaccgcgtatctg

cagtggagcagcctgaaagcgagcgataccgcgatgtatttttgcgcgcgcttttttggc

catattcgcgaaacccatggcgtgcagtggggccagggcaccatggtgaccgtgagcagc。

Preferably, the BDNF gene is optimized based on GENBANK BAO 51936.1.

Preferably, the specific steps of the preparation method of the anti-S1 PR5 nanobody are as follows:

(1) Immunization program

The alpaca is immunized by taking the full-length protein of S1PR5 (GenBank: AAH 67781.1) as an antigen, the total antigen amount of each immunization is kept between 1 and 2mg, the volume is less than 2mL, and the antigen and the adjuvant are emulsified 1:1 to form a uniform mixture before immunization, and the mixture is preserved at 4 ℃. And then recording blank alpaca numbers to start an immunization experiment, injecting at the left side and the right side near the alpaca neck lymph nodes each time, injecting at 2 points each time, injecting about 0.4mL emulsified antigen at each point, observing for half an hour after immunization to confirm that the alpaca is in a good state, and performing immunization once every 2 weeks for at least 4 times.

(2) Antibody Gene harvesting

Blood was collected from alpaca neck veins at 5-7 day intervals after the 4 th immunization for 50mL. Separating lymphocyte, extracting RNA, reverse transcribing to cDNA, designing primer according to the heavy chain antibody conserved region, and PCR amplifying to obtain heavy chain antibody variable region gene. The upstream primer is 5 'cggcggaagtgaaaaaaaaaaa3' and the downstream primer is 5'gccatgggtttcgcgaatat3'. The PCR system was 2. Mu.L of cDNA, 2. Mu.L of each of the upstream and downstream primers, 0.25. Mu.L of Taq DNA Polymerase Hot Start. Mu.L of enzyme, 5. Mu.L of 10 XTaq Buffer, 4. Mu.L of dNTP, and sterile water was made up to 50. Mu.L. The reaction procedure was pre-denaturation at 98℃for 3 min, cyclic amplification at 95℃for 30 sec, 57℃for 30 sec, 68℃for 40 sec, 2 sec increase per cycle, 22 cycles repeated, and annealing delay at 68℃for 5 min. Finally, the bands were visualized by agarose gel electrophoresis.

(3) Phage library construction and screening

The correctly amplified antibody gene fragment is connected to phagemid to construct phage display library. Then, phage clone capable of specifically binding to target antigen is obtained from the library through multiple rounds of screening by utilizing the principle of specific binding of antigen and antibody displayed on phage surface through phage display screening technology, so that the corresponding nano antibody gene is obtained, and the nucleotide sequence of the nano antibody gene is shown as SEQ ID No. 4.

(4) Expression and purification

The nanobody genes obtained by screening are cloned to a proper expression vector (plasmid vector) and then transformed into host cells for expression. The expressed nanobody can be purified by an affinity chromatography method.

In another aspect of the present invention, there is provided a method for preparing human bone marrow mesenchymal stem cells, comprising the steps of:

S1, preparing human bone marrow mesenchymal stem cells, namely taking bone marrow tissues containing primary mesenchymal stem cells, shearing small blocks, cleaning the bone marrow tissues with phosphate buffer solution on a culture dish until the washing solution is transparent, fully shearing the bone marrow tissues, transferring the sheared tissues to a 50ml centrifuge tube, adding 25ml of collagenase IV with the mass/volume concentration of 0.1%, shaking and digesting the collagen IV in a 37 ℃ incubator for 30min, adding 20ml of phosphate buffer solution into the digested tissues, uniformly mixing, filtering the mixture through a 100 mu m screen, centrifuging the filtrate in a 4 ℃ centrifuge for 5min at 1200 revolutions per min, removing supernatant to reserve cell sediment, adding 5ml of DMEM culture medium of 20% fetal bovine serum into the cell sediment, uniformly mixing, inoculating the cell sediment into a cell bottle, culturing the cell sediment in 37 ℃ and 5% CO ₂%, and carrying out passage every 2 days;

S2, constructing a lentiviral vector, directly synthesizing BDNF and anti-S1 PR5 nanobody nucleotide sequences according to the nucleotide sequences of SEQ ID No.3 and SEQ ID No.4, connecting into an enzyme-digested lentiviral expression vector pEF1 alpha, constructing a recombinant plasmid, and transforming DH5 alpha competent cells for amplification. Extracting plasmid, sequencing and verifying, preserving and constructing successfully plasmid, digesting 293T cells in logarithmic phase to prepare cell suspension, inoculating the cell suspension to a 24-hole culture plate, culturing in a cell culture box until the cell confluency is about 70-80%, co-transfecting the 293T cells with plasmid DNA, packaging plasmid psPAX vector, envelope plasmid pMD2G vector and LIPOFECTAMINE 3000, culturing for 6h, replacing fresh culture medium, continuously culturing for 72h, collecting 293T cell supernatant rich in slow virus, filtering, centrifuging and concentrating to obtain high-titer virus concentrate;

S3, taking BMSCs within 5 generations in the logarithmic growth phase, inoculating 100000 cells/hole into 12 culture plates, and 1ml of culture medium per hole. The medium was removed when the cell fusion was about 50%, and the virus concentrate of step S2 was transfected with PBS for 10 hours. After transfection, the medium is replaced to continue culturing and passaging. Screening for 7d by adopting a culture medium containing puromycin, and changing liquid once every 2d to obtain the BMSCs for stably expressing BDNF and the anti-S1 PR5 nano antibody.

In another aspect, the invention provides an application of the human bone marrow mesenchymal stem cells, in particular to an application of the human bone marrow mesenchymal stem cells in treating nervous system diseases, especially multiple sclerosis, and BDNF and anti-S1 PR5 nano-antibodies are modified into the human bone marrow mesenchymal stem cells, so that the multiple sclerosis can be effectively treated.

Compared with the prior art, the invention has the beneficial effects that:

According to the invention, BDNF and the anti-S1 PR5 nano antibody are modified to human bone marrow mesenchymal stem cells for the first time, wherein BDNF can promote proliferation and activity of human bone marrow mesenchymal stem cells, and the anti-S1 PR5 nano antibody is combined with S1PR5, so that survival of neurons or oligodendrocytes is promoted, apoptosis is inhibited, myelination is promoted, and the effect of treating multiple sclerosis is improved.

Drawings

FIG. 1 preparation flow of conventional nanobody

FIG. 2 shows a WB map of beta.3-tubulin and Olig2 protein expression

Detailed Description

The invention will be further illustrated with reference to specific examples, but the invention is not limited to these examples.

Example 1 preparation of anti-S1 PR5 nanobodies

(1) Immunization program

(2) Antibody Gene harvesting

(3) Phage library construction and screening

(4) Expression and purification

Example 2 BDNF and preparation of anti-S1 PR5 nanobody modified mesenchymal Stem cells

S1, preparing original human bone marrow mesenchymal stem cells, namely taking bone marrow tissues containing primary mesenchymal stem cells, shearing small blocks, cleaning the bone marrow tissues with phosphate buffer solution on a culture dish until the washing solution is transparent, fully shearing the bone marrow tissues, transferring the sheared tissues to a 50ml centrifuge tube, adding 25ml of collagenase IV with the mass/volume concentration of 0.1%, shaking and digesting the collagen IV in a 37 ℃ incubator for 30min, adding 20ml of phosphate buffer solution into the digested tissues, filtering the mixture through a 100 mu m screen after mixing the mixture, centrifuging the filtrate in a 4 ℃ centrifuge for 5min at 1200 revolutions per min, removing supernatant and reserving cell sediment, adding 5ml of DMEM culture medium of 20% fetal bovine serum into the cell sediment, uniformly mixing the DMEM culture medium, inoculating the DMEM culture medium into a cell bottle, culturing the cell bottle in 37 ℃ and 5% CO ₂%, and transferring the culture solution once every 2 days for passage;

S2, constructing a lentiviral vector, namely directly synthesizing BDNF and anti-S1 PR5 nanobody nucleotide sequences according to the nucleotide sequences of SEQ ID No.3 and SEQ ID No.4, connecting into an enzyme-digested lentiviral expression vector pEF1 alpha, constructing a recombinant plasmid, and transforming DH5 alpha competent cells for amplification. Extracting plasmid, sequencing and verifying, preserving and constructing successfully plasmid, digesting 293T cells in logarithmic phase to prepare cell suspension, inoculating the cell suspension to a 24-hole culture plate, culturing in a cell culture box until the cell confluency is about 70-80%, co-transfecting the 293T cells with plasmid DNA, packaging plasmid psPAX vector, envelope plasmid pMD2G vector and LIPOFECTAMINE 3000, culturing for 6h, replacing fresh culture medium, continuously culturing for 72h, collecting 293T cell supernatant rich in slow virus, filtering, centrifuging and concentrating to obtain high-titer virus concentrate;

S3, preparing genetically modified mesenchymal stem cells, namely taking BMSCs within 5 generations in the logarithmic growth phase, inoculating 100000 cells/hole into a 12-culture plate, and 1ml of culture medium is used for each hole. The medium was removed when the cell fusion was about 50%, and the virus concentrate of step S2 was transfected with PBS for 10 hours. Culturing by replacing culture medium, and passaging by conventional method. Screening for 7d by adopting a culture medium containing puromycin, and changing liquid once every 2d to obtain the BMSCs for stably expressing BDNF and the anti-S1 PR5 nano antibody.

Preparation of comparative example 1 BDNF modified mesenchymal Stem cells

The difference from example 1 is that the recombinant plasmid was constructed by introducing only the BDNF gene into the plasmid when constructing the lentiviral vector, and the remaining steps were the same.

Comparative example 2 preparation of anti-S1 PR5 nanobody modified mesenchymal Stem cells

The difference from example 1 is that only the gene of the anti-S1 PR5 nanobody was introduced into the plasmid to construct a recombinant plasmid when constructing a lentiviral vector, and the remaining steps are the same.

EXAMPLE 3 treatment of multiple sclerosis Using human mesenchymal Stem cells

1. Multiple sclerosis animal model construction

Modeling was performed using an Experimental Autoimmune Encephalomyelitis (EAE) model with similar pathological features to multiple sclerosis. The method comprises the following specific steps:

SPF-grade female mice C57BL/6 were selected for 10-14 weeks and divided into 4 groups of 6 mice each. The treatment groups are group A (model control group), group B (model+BDNF modified human bone marrow mesenchymal stem cell treatment group), group C (model+S1PR 5 nanobody modified human bone marrow mesenchymal stem cell treatment group) and group D (model+BDNF and S1PR5 nanobody double modified human bone marrow mesenchymal stem cell treatment group). MOG (myelin oligodendrocyte glycoprotein) 35-55 polypeptide was used as antigen, mixed with complete Freund's adjuvant, and injected intraperitoneally subcutaneously with 200. Mu.g each. After 7-14 days of injection, the modeling is successful because of dyskinesia, quadriplegia, paresthesia and the like.

2. Total protein and behavioral scores were collected after treatment with drug

The administration treatment was performed 14 days after the appearance of symptoms by injection. Dosing regimen was as follows, once daily per mouse, with each injection of 1mL (10 ⁵ cells/mL) of BMSCs by intraperitoneal administration for 14 days.

After 14 days of administration, 3 spinal cords were taken from each group for protein extraction, and Western Blot detection was performed on the neuronal marker protein beta 3-tubulin and the oligodendrocyte marker protein Olig 2.

After 14 days of administration, 3 groups were scored for behavior and the treatment effect was verified.

3. Evaluation of Effect

The WB detection shows that the protein expression of the beta 3-tubulin and the Olig2 of the group D is higher than that of the other groups, the group B shows higher protein expression of the beta 3-tubulin relative to the group A and the group C due to the modification of the brain-derived neurotrophic factor, and the group C shows higher protein expression of the Olig2 relative to the group A and the group B due to the modification of the anti-S1 PR5 nano antibody.

From the behavioral scores (table 1), the group D had better health and behavioral activity than the other groups, the group a had the worst health and behavioral activity, and the groups B and C exhibited some limited behavioral activity.

TABLE 1

Remarks are 0:no symptom, 1:tail weakness or paralysis, 2:rear limb weakness but normal walking, 3:rear limb paralysis, 4:four limbs paralysis, incapacity of autonomous movement, 5:death

Conclusion(s)

BMSCs modified by BDNF and anti-S1 PR5 nano antibodies treat multiple sclerosis, not only improve the activity of human bone marrow mesenchymal stem cells, but also promote the survival of neurons or oligodendrocytes, inhibit apoptosis, promote remyelination, and effectively treat multiple sclerosis.

The foregoing has outlined the basic principles, features, and advantages of the present invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, and that the above embodiments and descriptions are merely illustrative of the principles of the present invention, and various changes and modifications may be made without departing from the spirit and scope of the invention, which is defined in the appended claims. The scope of the invention is defined by the appended claims and equivalents thereof.

Claims

1. A genetically modified human bone marrow mesenchymal stem cell, characterized in that the human bone marrow mesenchymal stem cell is obtained by dual modification with brain-derived neurotrophic factor (BDNF) and anti-S1PR5 nanobody, wherein the amino acid sequence of BDNF is as shown in SEQ ID No. 1, and the amino acid sequence of the anti-S1PR5 nanobody is as shown in SEQ ID No. 1. As shown in No. 2, the human bone marrow mesenchymal stem cells were obtained by the following method: Bone marrow tissue containing primary mesenchymal stem cells was taken, cut into small pieces, washed with phosphate buffer on a culture dish until the washing solution became clear, and then thoroughly minced; the minced tissue was transferred to a 50ml centrifuge tube, 25ml of collagenase IV with a mass/volume concentration of 0.1% was added, and the mixture was placed in a 37℃ incubator and shaken for 30min for digestion; 20ml of phosphate buffer was added to the digested tissue, mixed well, and then filtered through a 100μm sieve. The filtrate was centrifuged at 1200 rpm for 5min at 4℃, the supernatant was discarded, and the cell pellet was retained; 5ml of DMEM medium containing 20% fetal bovine serum was added to the cell pellet, mixed well, and then seeded into a cell culture flask and cultured at 37℃ in 5% _CO2 . The medium was changed every 2 days for passage.

2. The human bone marrow mesenchymal stem cells according to claim 1, wherein the BDNF protein is obtained by optimization based on GENBANK number BAO51936.1.

3. The human bone marrow mesenchymal stem cells according to claim 1, characterized in that the method for preparing the anti-S1PR5 nanobody is as follows:

(1) Immunization schedule

Alpaca were immunized using the full-length S1PR5 protein (GenBank: AAH67781.1) as the antigen. The total amount of antigen for each immunization was kept 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 start the immunization experiment. Each time, 0.4 mL of emulsified antigen was injected into the left and right sides near the lymph nodes in the neck of the alpaca, with two injection points on each side. 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 total of at least 4 immunizations.

(2) Antibody gene acquisition

Five to seven days after the fourth immunization, 50 mL of blood was collected from the jugular vein of the alpaca. 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. The upstream primer was 5'cggcgcggaagtgaaaaa3', and the downstream primer was 5'gccatgggtttcgcgaatat3'. The PCR system consisted of 2 μL of cDNA, 2 μL each of the upstream and downstream primers, 0.25 μL of Taq DNA Polymerase Hot Start enzyme, 5 μL of 10X Taq Buffer, 4 μL of 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 3 seconds. 40 seconds, increasing by 2 seconds for each cycle, repeated for 22 cycles; Annealing extension: 68℃ for 5 minutes; Finally, observe the bands by agarose gel electrophoresis;

(3) Construction and screening of phage libraries

The correctly amplified antibody gene fragment is 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, after multiple rounds of screening, phage clones that can specifically bind to the target antigen are screened from the library, thereby obtaining the corresponding nanobody gene, whose nucleotide sequence is shown in SEQ ID No. 4.

(4) Expression and purification

The selected nanobody genes were cloned into a suitable expression vector and then transformed into host cells for expression. The expressed nanobody was then purified by affinity chromatography.

4. A method for preparing gene-modified human bone marrow mesenchymal stem cells as described in claim 1, characterized in that the method comprises the following steps:

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% by volume) 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, 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, and seeded into cell culture flasks. The cells were cultured at 37°C in 5% _CO2 , with the medium changed every 2 days for passage.

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 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 culture 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 virus concentrate.

S3. Take BMSCs in the logarithmic growth phase (generation 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 reaches 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. Use puromycin-containing culture medium for selection for 7 days, changing the medium every 2 days to obtain BMSCs that stably express BDNF and anti-S1PR5 nanobodies.

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

6. The use of the human bone marrow mesenchymal stem cells according to claim 1 in the preparation of a drug for treating nervous system diseases, wherein the nervous system disease is multiple sclerosis.

7. The application according to claim 6, characterized in that the human bone marrow mesenchymal stem cells promote the survival of neurons and oligodendrocytes, inhibit cell apoptosis, and promote myelin regeneration.