CN106011173B

CN106011173B - Preparation method of human oligodendrocyte progenitor cells for inhibiting nerve secondary injury, kit and application thereof

Info

Publication number: CN106011173B
Application number: CN201610034981.0A
Authority: CN
Inventors: 黄启明; 王旭; 王玉东
Original assignee: Jiangxi Austria Biological Technology Co ltd
Current assignee: Jiangxi Austria Biological Technology Co. Ltd.
Priority date: 2016-01-20
Filing date: 2016-01-20
Publication date: 2020-08-11
Anticipated expiration: 2036-01-20
Also published as: CN106011173A

Abstract

The invention provides a preparation method of human oligodendrocyte progenitor cells for inhibiting secondary damage of nerves, wherein the method comprises the steps of constructing virus expression vectors of two glial growth factors, co-transfecting the human oligodendrocyte progenitor cells, highly expressing the two glial growth factors, namely growth-related factor 43 and glial growth factor 2, playing the combined action of the two factors to promote the repair of the oligodendrocyte and inhibit the hyperplastic reaction and glial scar formation after the oligodendrocyte is damaged so as to cause the secondary damage of the nerves; meanwhile, the oligodendrocyte progenitor cells have self proliferation and differentiation capacity, participate in the repair of nerve functions and have no tumorigenesis; and provides a kit for preparing the human oligodendrocyte precursor cells for inhibiting the secondary damage of the nerves. The human oligodendrocyte precursor cells provided by the invention have good treatment effect on nervous system diseases.

Description

Preparation method of human oligodendrocyte progenitor cells for inhibiting nerve secondary injury, kit and application thereof

Technical Field

The invention relates to a preparation method of a human oligodendrocyte progenitor cell for inhibiting the secondary damage of nerves and a human oligodendrocyte progenitor cell for inhibiting the secondary damage of nerves obtained by the method, in particular to a virus expression vector containing two glial growth factors, which is constructed to ensure that the human oligodendrocyte progenitor cell can stably express the two glial growth factors and jointly exert the biological functions of the two glial growth factors; and provides a kit for preparing the human oligodendrocyte precursor cells for inhibiting the secondary damage of the nerves. The human oligodendrocyte progenitor cells provided by the invention maintain good proliferation capacity, participate in the repair of nerve functions, have no tumorigenicity, and are expected to be used for clinically treating nervous system diseases and promoting the repair of damaged nerve cells.

Background

Under the conditions of ischemia, hypoxia, injury and the like, the nervous system diseases cause obvious change of calcium ion concentration after astrocyte injury, activate intracellular calcium ion signal conduction and participate in proliferative reaction after astrocyte injury, and the abnormal proliferation of astrocytes causes the formation of glial scars, thereby preventing the effective regeneration and growth of injured nerve axons, and mainly being central nerve injury diseases, namely traumatic brain injury, cerebral hemorrhage, cerebral ischemia, spinal cord injury and the like.

Astrocytes in early stage of nerve injury are in early stage of maturation, can secrete cytokines to maintain and promote the function of neurons, and play a role in neural tissue scaffolds; however, after the growth of the axon, various beneficial functions in the early stage gradually disappear, and instead, harmful factors are secreted to form a chemical glial barrier, so that the nerve regeneration is influenced, and the axon elongation is hindered. Mature astrocytes located within the lesion interface show proteoglycan expression that inhibits axonal growth and play a major role in scar tissue formation. Glial hyperproliferation and glial scarring, which lead to mechanical disturbances, affect axonal regeneration, elongation and fusion, and may form microvascular sleeves, compress microvessels, affect local blood supply, all of which have a secondary damaging effect on nerve cells.

The applicant of the invention unexpectedly finds that the glial growth factor secreted by the adult stem cell plays an important role in inhibiting the secondary damage of nerves, can improve the microenvironment of an organism and promote the repair effect of stem cells and endogenous stem cells, but the glial growth factor secreted by the stem cell is very limited, and researches suggest that the glial growth factor secreted by the adult stem cell actually plays a role in stem cell transplantation treatment.

The invention recombines the constructed segment containing two glia growth factors on a virus vector, expresses the two glia growth factors after co-transfecting the human oligodendrocyte progenitor cells, and exerts the biological functions of the two glia growth factors together without tumorigenicity, thereby having good synergistic effect. The human oligodendrocyte progenitor cells are convenient to source and mainly comprise cord blood, placental blood, bone marrow, aborted fetal brain and the like, the cell culture is simple to obtain, individuation can be realized, and allogeneic cells with low immunogenicity can also be used. Therefore, the human oligodendrocyte progenitor cells expressing the two glia growth factors are simple to prepare, highly express the two glia growth factors, inhibit the abnormal proliferation of astrocytes and the formation of glia scars, jointly create a microenvironment beneficial to nerve regeneration, inhibit the secondary damage of nerves, and are expected to produce better treatment effect on central injury diseases.

Disclosure of Invention

In recent years, the clinical curative effect of the stem cells on treating nervous system diseases is widely accepted, and the stem cell transplantation promotes the repair and the regeneration of nerve cells and improves the recovery of axon and behavior function of nerves. Astrocyte maturation and abnormal proliferation are caused by microglial activation in the later stage of central nervous injury diseases, which leads to glial scar formation and neuroinflammatory reaction, and secondary injury is caused to nerve cells.

Based on the earlier research work basis, the invention utilizes the characteristics of proliferation differentiation and 'homing' of human oligodendrocyte progenitor cells to transfect recombinant viral vectors of two constructed glial growth factor gene segments, can highly express the two glial growth factors, synergistically play the biological action of the glial growth factors and the functions of the oligodendrocyte progenitor cells, fill damaged interfaces, inhibit the formation of glial scars, rearrange receptor tissues, delay the expression of axon growth inhibition factor proteoglycan, and inhibit secondary damage of nerves.

Wherein the glia growth factor is growth-associated factor 43(GAP-43) and glia growth factor 2 (GGF-2); GAP-43 expression up-regulation promotes neuron growth and inhibits glial scar formation; the increase of GGF-2 expression is helpful for recovering nerve function, improving nerve inflammatory reaction and controlling damage to nerve cells. The two ingredients have synergistic effect, and can fill damaged interface, inhibit colloid scar formation, rearrange receptor tissue, delay the expression of axon growth inhibitory factor proteoglycan, and inhibit nerve secondary damage.

In the invention, through research and development work, human Oligodendrocyte Progenitor Cells (OPCs) transfected by a glial growth factor lentiviral vector are found, and the glial growth factor stably expressed by the human Oligodendrocyte progenitor cells can inhibit the secondary damage of nerves and has no tumorigenic activity. The research result indicates that the human oligodendrocyte progenitor cells can be transfected by the lentiviral vector constructed by the two glial growth factors, so that the human oligodendrocyte progenitor cells can still stably express the two glial growth factors after multiple subcultures, and have the capacity of exerting respective biological functions. The cell number used in clinical treatment is ensured, and simultaneously, the strong proliferation, differentiation and homing activities of stem cells are also kept, and the cell plays a better role in clinical treatment of central nerve injury diseases.

The invention provides a preparation method of human oligodendrocyte progenitor cells for inhibiting secondary damage of nerves, which is characterized by comprising the steps of constructing virus expression vectors of two glial growth factors, co-transfecting the human oligodendrocyte progenitor cells to prepare the oligodendrocyte progenitor cells capable of highly expressing the two glial growth factors, and inhibiting the secondary damage of the nerves caused by glial cell proliferation to promote the repair of the nerve damage; wherein the glial growth factor is growth-related factor 43(GAP-43) and glial growth factor 2 (GGF-2).

After the OPCs transfected with recombinant GAP-43 and GGF-2 virus vectors are subcultured to 10 th generation through a fluorescent quantitative PCR kit according to an instruction, the expression GAP-43 and GGF-2 genes are higher than those of untransfected 1 st generation OPCs by at least 45 times and 67 times respectively;

in a rat model with spinal cord injury, the motor function score improvement value of BBB (BassoBeattie Bresnahan) 28 days after the transplantation of OPCs cotransfected by GAP43/GGF2 virus is 20.00 +/-5.00, and the amplitude improvement value of cortical somatosensory evoked potential CSEP is 6.00 +/-1.00; the GAP43/GGF2-OPCs inhibit astrocyte proliferation and glial scarring in the treatment of spinal cord injured rats.

The human oligodendrocyte precursor cells are convenient to obtain, simple to obtain by cell culture, can be obtained from bone marrow of a patient in an individualized way, can also be produced in a large scale as variant, and can be applied in an individualized way. The human oligodendrocyte precursor cells are derived from human waste umbilical cord blood, placental blood, autologous bone marrow, aborted fetal brain, and the like, and preferably, are derived from human autologous bone marrow.

The invention relates to a method for preparing human oligodendrocyte progenitor cells, namely, the human oligodendrocyte progenitor cells are prepared by carrying out lymphopoiesis on bone marrowSeparating and purifying the obtained mononuclear cells from the cell separation medium, coating with polylysine at 10-200 μ g/ml in 75cm serum-free oligodendrocyte medium (i.e. DMEM/F12 (1: 1) containing 1% N2, 2mM L-glutamine, 5-100ng/ml basic fibroblast growth factor, 1-50ng/ml fibroblast growth factor 4, 1-100ng/ml stem cell growth factor and 1-100ng/ml FMS-like tyrosine kinase 3 ligand)²Carrying out primary and subculture in a cell culture bottle to obtain human oligodendrocyte progenitor cells; preferably, the serum-free oligodendrocyte medium is DMEM/F12 (1: 1) containing 1% N2, 2mM L-glutamine, 30ng/ml basic fibroblast growth factor, 5ng/ml fibroblast growth factor 4, 10ng/ml stem cell growth factor and 5ng/ml FMS-like tyrosine kinase 3 ligand; 75cm coated with 50. mu.g/ml polylysine²A cell culture flask.

The human oligodendrocyte precursor cells are detected by a flow cytometry technology, and OPCs markers A2B5, O4 and Sox10 are all expressed by more than 95%.

The virus vector can stably proliferate and express target genes in cells, mainly comprises lentiviruses, adenoviruses, retroviruses and the like, and preferably selects a lentivirus expression system.

The invention provides a preparation method of human oligodendrocyte precursor cells for inhibiting nerve secondary injury, which is characterized in that human GAP-43 and GGF-2 sequences are amplified from normal human peripheral blood genome DNA through PCR to obtain GAP-43 and GGF-2 target fragments. After the two target DNA fragments are connected to an expression plasmid to construct a recombinant plasmid, the constructed plasmid can be stored in an ultra-low temperature refrigerator at minus 80 ℃ after positive cloning PCR identification and sequencing identification. And cloning GAP-43 and GGF-2 gene segments onto a lentiviral vector, packaging the lentiviral vector into recombinant GAP-43 and GGF-2 lentiviral vectors, co-transfecting human oligodendrocyte progenitor cells with the lentiviral vector, and preparing the human oligodendrocyte progenitor cells transfected by GAP43/GGF2 lentiviruses.

Observing the luminescence condition of Green Fluorescent Protein (GFP) under a fluorescence microscope 72 hours after transfection, wherein the GFP color development of the oligodendrocyte precursor cells exceeds more than 90 percent, which indicates that the transfection of GAP-43 and GGF-2 lentiviruses to the human oligodendrocyte precursor cells is successful, and the human oligodendrocyte precursor cells which continuously express GAP-43 and GGF-2 are obtained;

after GAP-43 and GGF-2 lentivirus transfected human oligodendrocyte progenitor cells are subcultured to 10 th generation, the expression GAP-43 and GGF-2 genes of the cells are respectively higher than 45.89 and 67.98 times of untransfected OPCs by a fluorescent quantitative PCR kit according to the instruction.

The human oligodendrocyte precursor cells for inhibiting the secondary damage of nerves can be subjected to in vitro subculture, namely OPCs transfected by GAP-43 and GGF-2 lentiviral vectors are subjected to in vitro subculture by a complete culture solution DMEM/F12, when the cells are cultured for 5 to 6 days, the cells grow and fuse to more than 90 percent, and pancreatin with the mass-volume ratio of 0.25 percent is required for digestion and subculture;

compared with OPCs which are not transfected with GAP-43 and GGF-2 dual-lentiviral vectors, the human oligodendrocyte progenitor cells have stronger proliferation speed, have the proliferation times of more than 500 times after subculture to the 10 th generation, and have the capacity of differentiating into oligodendrocytes.

After the human oligodendrocyte precursor cells are subcultured for 10 generations, the human oligodendrocyte precursor cells still highly express growth-related factors 43 and glial growth factor 2, and have similar astrocyte morphology compared with OPCs which are not transfected with GAP-43 and GGF-2 dual lentiviral vectors, and can differentiate the oligodendrocytes.

In a rat model with spinal cord injury, the BBB motor function score and cortical somatosensory evoked potential improvement after transplantation of OPCs co-transfected by GAP43/GGF2 lentivirus are obviously higher than those of untransfected OPCs, so that the nerve function recovery of the rat with spinal cord injury is promoted; glial fibrillary acidic protein immunohistochemistry shows that compared with the transplantation of the untransfected OPCs, the OPCs cotransfected by GAP43/GGF2 lentivirus have obviously reduced positive cell expression quantity and obviously reduced glial scar range.

The tumor formation phenomenon is not found in-vivo and in-vitro tumor formation experiments, and the safety of the in-vivo and in-vitro tumor formation experiments is ensured.

The invention also provides a prepared kit for inhibiting the human oligodendrocyte precursor cells of the nerve secondary injury, which is characterized by comprising:

1) basal medium for human oligodendrocyte progenitor cells;

2) packaged growth-related factor 43 and glial growth factor 2 high-expression virus;

3) an enzyme that digests the cells;

4) a cytokine and;

5) instructions for use;

wherein the instructions for use comprise the methods described above.

The invention also provides application of the human oligodendrocyte precursor cells for inhibiting the secondary damage of the nerve, ensures that a sufficient number of human oligodendrocyte precursor cells are obtained by in vitro culture, has the advantages of stably expressing two glial growth factors, exerting the biological functions of the two glial growth factors together, promoting the regeneration of axons of nerves and protecting the physiological functions of the nerves, effectively inducing the new astrocytes to be linearly arranged, providing an ideal channel for the regeneration and the growth of the axons of the nerves, and regulating and controlling the excessive proliferation of the astrocytes and the formation of glial scars, thereby inhibiting the secondary damage to the nerves.

Drawings

FIG. 1 is a flow chart of flow cytometric assay of human oligodendrocyte precursor cells prepared in example 2

FIG. 2 is a graph showing immunofluorescence of recombinant GAP-43 and GGF-2 lentiviruses after transfection of OPCs

FIG. 3 is a graph showing the gene expression level of recombinant GAP-43 and GGF-2 lentivirus of generation 10 after co-transfection of OPCs

FIG. 4 shows the immunofluorescence chart of differentiation of OPCs co-transfected with 10 th generation recombinant GAP43/GGF2 lentivirus into oligodendrocytes

FIG. 5 is a graph showing the results of improvement of GFAP expression and glial scar after transplantation of rat model GAP43/GGF2-OPCs for spinal cord injury

Detailed Description

The invention provides a preparation method of human oligodendrocyte progenitor cells for inhibiting secondary damage of nerves, which is characterized by comprising the steps of constructing virus expression vectors of two glial growth factors, co-transfecting the human oligodendrocyte progenitor cells to prepare the oligodendrocyte progenitor cells capable of highly expressing the two glial growth factors, inhibiting the proliferation of glial cells to cause the secondary damage of the nerves and promoting the repair of the nerve damage.

The human oligodendrocyte precursor cells are convenient to source, can be obtained from human waste umbilical cord blood, placental blood, autologous bone marrow, aborted fetal brain and the like, and preferably are derived from human autologous bone marrow.

The preparation method of human oligodendrocyte progenitor cells comprises the steps of taking mononuclear cells obtained by separating and purifying 20ml of bone marrow through lymphocyte separating medium, and re-suspending the mononuclear cells to 2-5 × 10 by using a serum-free oligodendrocyte culture medium (namely DMEM/F12 (1: 1) containing 1% of N2, 2 mML-glutamine, 5-100ng/ml of basic fibroblast growth factor, 1-50ng/ml of fibroblast growth factor 4, 1-100ng/ml of stem cell growth factor and 1-100ng/ml of FMS-like tyrosine kinase 3 ligand)⁶Sucking 10ml of the solution and adding the solution into a polylysine-coated 75cm solution of 10-200. mu.g/ml²In a cell culture flask, 5% CO at 37 ℃₂The cells were cultured in a cell incubator for 48 hours. Replacing a fresh serum-free oligodendrocyte medium containing the cell factors, continuously culturing until the growth and fusion of the cells reach about 90 percent, digesting by using 0.25 percent (mass-volume ratio) pancreatin (containing 0.02 percent of EDTA) by mass-volume ratio, carrying out subculture, namely, carrying out 1 bottle passage and 2 bottles, supplementing the fresh serum-free oligodendrocyte medium containing the cell factors, and continuously culturing to obtain the human oligodendrocyte progenitor cells.

Preferably, the serum-free oligodendrocyte medium is DMEM/F12 (1: 1) containing 1% N2, 2mM L-glutamine, 30ng/ml basic fibroblast growth factor, 5ng/ml basic fibroblast growth factor 4, 10ng/ml stem cell growth factor and 5ng/ml FMS-like tyrosine kinase 3 ligand; 75cm coated with 50. mu.g/ml polylysine²A cell culture flask.

The gene segments of the two glia growth factors are amplified and recombined on expression plasmids by a PCR technology, then are recombined on a virus expression vector, and are packaged into 293T cells to prepare the virus with high expression target genes.

The glial growth factor for inhibiting the secondary damage of nerves is growth-related factor 43(GAP-43) and glial growth factor 2 (GGF-2).

The virus vector can stably proliferate and express target genes in cells, mainly comprises lentivirus, adenovirus, retrovirus and the like, preferably selects lentivirus expression systems, is a commercial product and can be purchased from commercial companies.

Amplifying human GAP-43 and GGF-2 sequences from normal human peripheral blood genome DNA by PCR, preparing clone connecting liquid by connecting GAP-43 and GGF-2 target fragments and a pET28a vector fragment subjected to XhoI/Nde I double enzyme digestion respectively at 4 ℃ for 12 hours under the action of T4DNA ligase, and carrying out positive clone PCR identification and sequencing identification after transforming escherichia coli competent cells DH5 a; after the size and the sequence of the PCR product gel electrophoresis detection and sequencing identification accord with GAP-43 and GGF-2, transferring the bacterial liquid with correct sequencing into 10ml LB liquid culture medium containing corresponding antibiotics, culturing overnight at 37 ℃, performing plasmid extraction by using a Beijing Tiangen organism endotoxin-free plasmid small-medium-volume extraction kit, and storing the qualified recombinant plasmid in an ultra-low temperature refrigerator at-80 ℃ for a long time; the sizes of the GAP-43 and the GGF-2 are respectively about 706bp and 1268 bp;

carrying out XhoI/Nde I double enzyme digestion on GAP-43, GGF-2DNA fragments and GV358 vectors respectively, carrying out ligation reaction on the GAP-43 fragments, the GV358 vectors, the GGF-2 fragments and the GV358 vectors at 37 ℃ for 12 hours respectively under the action of T4DNA ligase to prepare clone ligation liquid, transforming escherichia coli competent cells DH5a, and then carrying out positive clone PCR identification and sequencing identification;

293T cells in a good cell state and in a logarithmic growth phase were collected, and after counting, the cells were cultured in 10cm dishes 6 × 10⁶The number of cells was plated in a petri dish at 37 ℃ with 5% CO₂Culturing in an incubator overnight; removing the culture solution before the next day of transfection, and replacing 5ml of Opti-MEM culture solution; adding 9 μ g of the packaged mixed solution and 3 μ g of the lentivirus expression plasmid into 1.5ml of Opti-MEM, gently mixing, adding 36 μ l of lipofectamine2000 into 1.5ml of Opti-MEM, gently mixing, and standing at room temperature for 5 min; mixed plasmid solution and lipofectamine2000 dilutionStanding at room temperature for 20 min; the mixed solution is slowly dripped into the 293T cell culture solution, mixed evenly and treated at 37 ℃ with 5% CO₂Culturing in a cell culture box; culturing for 6h, discarding the culture medium containing the transfection mixture, adding 10ml of PBS (phosphate buffer solution) for washing once, gently shaking the culture dish to wash the residual transfection mixture, and then pouring and discarding; slowly adding 10% serum-containing cell culture medium 20ml, and adding 5% CO at 37 deg.C₂Continuously culturing for 48-72h in the incubator;

collecting 293T cell supernatant 48h after transfection according to cell state; centrifuging at 4000g for 10min at 4 deg.C to remove cell debris; filtering the supernatant with a 0.45 μm filter in a 40ml ultracentrifuge tube; respectively balancing samples, putting ultracentrifuge tubes with virus supernatant into a Beckman ultracentrifuge one by one, setting the centrifugation parameters to be 25000rpm, setting the centrifugation time to be 2h, and controlling the centrifugation temperature to be 4 ℃; after the centrifugation is finished, removing the supernatant, removing the liquid remained on the tube wall as much as possible, adding a virus preservation solution, and lightly and repeatedly blowing and resuspending; after full dissolution, centrifuging at high speed of 10000rpm for 5min, taking supernatant to measure titer by a fluorescence method, subpackaging the packaged GAP-43 and GGF-2 lentivirus according to 50 mu l of 2E +8TU/ml, and storing in an ultra-low temperature refrigerator at-80 ℃;

adding 10 μ l of each of recombinant GAP-43 and GGF-2 lentivirus into 10-200 μ g/ml polylysine-coated six-well culture plate of human oligodendrocyte progenitor cells (2 ml for system), mixing, 37 deg.C, and 5% CO₂After incubation in the incubator of (1) for 8-12 hours, complete medium DMEM/F12 (1: 1) containing 1% N2, 2mM L-glutamine, 5-100ng/ml basic fibroblast growth factor, 1-50ng/ml fibroblast growth factor 4, 1-100ng/ml stem cell growth factor and 1-100ng/ml FMS-like tyrosine kinase 3 ligand is replaced; when the cell growth and fusion reach 90%, the cells are digested with 0.25% pancreatin and transferred to 25cm coated with 10-200. mu.g/ml polylysine²Growth in flasks, 1 well for 1 flask at 25cm²Continuing digestion subculture when the fusion in the culture flask reaches 90%; the growth transfections were divided into 3 groups: untransfected control group, blank lentivirus transfection group, co-transfected GAP-43 and GGF-2 lentivirus transfection group;

observing the luminescence condition of the green fluorescent protein under a fluorescence microscope for 72 hours after transfection, wherein the GFP color development of the oligodendrocyte progenitor cells exceeds more than 90 percent, which shows that the transfection of the human oligodendrocyte progenitor cells by the slow viruses GAP-43 and GGF-2 through cotransfection is successful, and the human oligodendrocyte progenitor cells which continuously express GAP-43 and GGF-2 are obtained;

After the human oligodendrocyte precursor cells for inhibiting the secondary damage of nerves are injected subcutaneously in a nude mouse body, no tumorigenesis phenomenon is found, the safety of the human oligodendrocyte precursor cells is ensured, and the inoculated breast cancer cell line MCF-7 positive control has tumorigenesis.

1) basal medium for human oligodendrocyte progenitor cells;

3) an enzyme that digests the cells;

4) a cytokine and;

5) instructions for use;

wherein the instructions for use comprise the methods described above.

The invention also provides the application of the human oligodendrocyte progenitor cells for inhibiting the secondary damage of nerves, the human oligodendrocyte progenitor cells for highly expressing GAP-43 and GGF-2 can fill the damaged interface, inhibit the formation of colloid scars, rearrange receptor tissues and delay the expression of axon growth inhibitory factor proteoglycan; thereby effectively improving the microenvironment for axon regeneration and promoting the growth of axons after acute spinal cord injury; inhibit secondary damage of nerve, i.e. inhibit atrophy of neuron after axon cutting and promote recovery of behavior ability remarkably.

Specific examples of the present invention will be described below, but the technical scope of the present invention is not limited to these examples.

Example 1 construction and packaging of recombinant GAP-43 and GGF-2 Lentiviral vectors

Amplifying human GAP-43 and GGF-2 sequences from normal human peripheral blood genomic DNA by PCR, performing ligation reaction of GAP-43 and GGF-2 target fragments with pET28a vector (Invitrogen) fragment double-digested with XhoI/Nde I (TAKARA, Japan) at 4 deg.C for 12 hr under the action of T4DNA ligase (TAKARA, Japan) to prepare clone ligation solution, transforming Escherichia coli competent cell DH5a (Invitrogen, USA), and performing positive clone PCR identification and sequencing identification; the sizes of the GAP-43 and GGF-2 gene fragments are respectively identified to be about 706bp and 1268bp, and the sizes and sequences are in accordance with the sizes and sequences of the GAP-43 and the GGF-2. Transferring the correctly sequenced bacterium liquid into 10ml LB liquid culture medium containing aminobenzene antibiotics, culturing overnight at 37 ℃, extracting plasmids by using a Beijing Tiangen biological endotoxin-free plasmid miniprep medium kit, and placing the qualified recombinant plasmids in an ultra-low temperature refrigerator at-80 ℃ for long-term storage.

Carrying out XhoI/Nde I double enzyme digestion on GAP-43, GGF-2DNA fragments and GV358 vectors (Shanghai Jikai), respectively, carrying out ligation reaction on the GAP-43 fragments, the GV358 vectors, the GGF-2 fragments and the GV358 vectors at 37 ℃ for 12 hours under the action of T4DNA ligase to prepare clone ligation solutions, transforming Escherichia coli competent cells DH5a, and carrying out positive clone PCR identification and sequencing identification.

293T cells (Invitrogen, USA) in a good cell state and a logarithmic growth phase were collected, and after counting, the cells were collected in 10cm dishes 6 × 10⁶The number of cells was plated in a petri dish at 37 ℃ with 5% CO₂Culturing in an incubator overnight; removing the culture solution before the next day of transfection, and replacing 5ml of Opti-MEM culture solution; adding 9 μ g of the packaged mixture and 3 μ g of the lentiviral expression plasmid into 1.5ml of Opti-MEM, gently mixing, adding 36 μ l of lipofectamine2000 (Invitrogen, USA) into 1.5ml of Opti-MEM, gently mixing, and standing at room temperature for 5 min; mixing the plasmid solution and lipofectamine2000 diluent, and standing at room temperature for 20 min; the mixed solution is slowly dripped into the 293T cell culture solution, mixed evenly and treated at 37 ℃ with 5% CO₂Culturing in a cell culture box; culturing for 6h, discarding the culture medium containing the transfection mixture, adding 10ml of PBS (phosphate buffer solution) for washing once, gently shaking the culture dish to wash the residual transfection mixture, and then pouring and discarding; slowly adding 10% serum-containing cell culture medium 20ml, and adding 5% CO at 37 deg.C₂Continuously culturing for 48-72h in the incubator;

EXAMPLE 2 preparation of human oligodendrocyte progenitor cells

Collecting 20ml bone marrow of donor volunteer, separating and purifying to obtain mononuclear cells with trypan blue count of 3 × 10⁷Each of these cells was resuspended to 3 × 10-10% using serum-free oligodendrocyte medium (i.e., DMEM/F12 (1: 1) (Gibco, USA) containing 1% N2, 2mM L-glutamine (Gibco, USA), 30ng/ml basic fibroblast growth factor, 5ng/ml fibroblast growth factor 4, 10ng/ml stem cell growth factor, and 5ng/ml FMS-like tyrosine kinase 3 ligand (PeproTech, USA))⁶Pipetting 10ml into 75cm of polylysine coated 50. mu.g/ml²In a cell culture flask, 5% CO at 37 ℃₂The cells were cultured in a cell incubator for 48 hours.

Replacing a fresh serum-free oligodendrocyte medium containing the cell factors after 48 hours of culture, continuously culturing, replacing the fresh serum-free oligodendrocyte medium containing the cell factors at intervals of 2 days until the cells grow and fuse to about 90 percent, digesting by using 0.25 percent (mass to volume) of pancreatin (containing 0.02 percent of EDTA) (Gibco company of America), carrying out subculture, namely 1 bottle of passage 2 bottles, supplementing the fresh serum-free oligodendrocyte medium containing the cell factors, and continuously culturing to obtain the human oligodendrocyte progenitor cells.

0.6 × 10 after trypan blue staining count⁶OPC cells were added in three groups, the first group containing 20. mu.L of mouse anti-human A2B5 monoclonal antibody (Millipore, USA), and secondary antibody goat anti-mouse IgM-FITC (Invitrogen, USA); 20 μ L of mouse anti-human O4 mab (Millipore, USA), secondary antibody goat anti-mouse IgM-PE (Invitrogen, USA); the second group was supplemented with 20. mu.L of mouse anti-human Sox10 mab (Millipore, USA) and secondary goat anti-mouse IgM-FITC (Invitrogen, USA); mu.L of rabbit anti-human GFAP monoclonal antibody (Millipore, USA),the second antibody goat anti-mouse IgG-PE (Invitrogen, USA) and the third group is isotype control, which are respectively added to 20. mu.L FITC labeled mouse IgM and 20. mu.L PE labeled mouse IgM, and the mixture is placed in a refrigerator at 4 ℃ for staining for 30 minutes, then washed three times with 1mL 1 × Phosphate Buffer Solution (PBS), and finally the washed cells are resuspended with 0.5mL 1 × PBS, and the obtained washed cells are detected by FACS Calibur flow cytometer (BD, USA). fig. 1 shows that the human oligodendrocyte progenitor cells are detected by flow cytometry, and the expression of OPCs markers A2B5, O4 and Sox10 are respectively 99.08%, 96.40% and 96.90%, and the expression of GFAP is 9.62%.

Example 3 preparation of recombinant GAP-43 and GGF-2 lentiviruses transfected human oligodendrocyte precursor cells

Adding 10 μ l of each of the recombinant GAP-43 and GGF-2 lentivirus 1E +7TU/ml into a 50 μ g/ml polylysine-coated six-well culture plate of human oligodendrocyte progenitor cells (2 ml), mixing, and adding 5% CO at 37 deg.C₂After incubation in the incubator of (1) for 8-12 hours, complete medium DMEM/F12 (1: 1) containing 1% N2, 2mM L-glutamine, 30ng/ml basic fibroblast growth factor, 5ng/ml fibroblast growth factor 4, 10ng/ml stem cell growth factor and 5ng/ml FMS-like tyrosine kinase 3 ligand was replaced; when the cells reached 90% confluence, they were digested with 0.25% trypsin and transferred to 25cm coated with 50. mu.g/ml polylysine²Growth in flasks, 1 well for 1 flask at 25cm²Continuing digestion subculture when the fusion in the culture flask reaches 90%; the growth transfections were divided into 3 groups: untransfected control group, blank lentivirus transfection group, co-transfected GAP-43 and GGF-2 lentivirus transfection group.

FIG. 2 is an immunofluorescence picture of recombinant GAP-43 and GGF-2 lentiviruses after transfection of OPCs. The recombinant GAP-43 and GGF-2 lentivirus vectors carry Green Fluorescent Protein (GFP), and OPCs transfected with the recombinant GAP-43 and GGF-2 lentiviruses are green under a fluorescence inverted microscope, which shows that the recombinant GAP-43 and GGF-2 lentivirus transfected OPCs are successfully constructed.

FIG. 3 shows the expression levels of GAP-43 and GGF-2 in the 10 th generation of OPCs transfected with recombinant GAP-43 and GGF-2 lentiviruses. After the OPCs transfected with recombinant GAP-43 and GGF-2 lentiviral vectors were subcultured to passage 10 as detected by a fluorescent quantitative PCR kit according to the instructions (Invitrogen, USA), the expression GAP-43 and GGF-2 genes were higher than those of untransfected passage 1 OPCs by 45.89 and 67.98 times, respectively.

Example 4 kit for inhibiting neural secondary injury of human oligodendrocyte precursor cells

The kit for inhibiting the secondary damage of the nerves of the human oligodendrocyte precursor cells comprises:

1) human oligodendrocyte progenitor cell basal medium, i.e., DMEM/F12 (1: 1);

3) enzyme for digesting cells, 0.25% (mass to volume) pancreatin (containing 0.02% by mass to volume of EDTA);

4) cytokines, namely 1% N2, 2mM L-glutamine, 5-100ng/ml basic fibroblast growth factor, 1-50ng/ml fibroblast growth factor 4, 1-100ng/ml stem cell growth factor and 1-100ng/ml FMS-like tyrosine kinase 3 ligand;

5) instructions for use;

wherein the instructions for use comprise the methods described in examples 1-3.

Example 5 differentiation of human oligodendrocyte progenitor cells into oligodendrocytes transfected with recombinant GAP43/GGF2 lentivirus

The 10 th generation of OPCs transfected with recombinant GAP43/GGF2 lentivirus were inoculated into 6-well plates previously loaded with 50. mu.g/ml polylysine-coated coverslips, and after the cells had grown to complete confluence, an induction solution (DMEM/F12 (1: 1) containing 10ng/ml neurotrophic factor 3 (PeproTech, USA) and 50ng/ml β -mercaptoethanol (Invitrogen, USA)) was used at 37 ℃ with 5% CO₂Culturing for 24h in incubator, removing inducing solution, adding completely differentiated culture solution DMEM/F12 (1: 1) (containing 2mM L-glutamine, 30ng/ml basic fibroblast growth factor, 5ng/ml fibroblast growth factor 4, 10ng/ml stem cell growth factor and 5ng/ml FMS-like tyrosine kinase 3 ligand and 10% fetal bovine serum), culturing at 37 deg.C and 5% CO₂Culturing in incubator, changing culture medium 1 time every 72h, and culturing for 7 days. Culturing for 7 days, fixing cells with 4% paraformaldehyde, and performing glial fibrillary acidSex protein (GFAP) immunocytochemical examination.

GAP43/GGF2-OPCs were fixed in 4% cold paraformaldehyde for 15 minutes and then protected from light. After the paraformaldehyde was aspirated, the column was washed three times with ice PBS for 5 minutes each. Cells were covered with 0.5% Triton X-100 for 10min and washed three times with ice PBS for 5min each. Import foetal sheep serum from the same host as the secondary antibody was blocked for 30 minutes at room temperature. Preparing a primary antibody: rabbit anti-human GFAP (Millipore, USA) is diluted 200 times with fetal sheep serum, primary antibody covering cells are added, and the cells are wrapped in tinfoil for 4 degrees and kept out of the sun overnight. The next day: the cells were removed and allowed to re-warm to room temperature for about 1 h. Washed twice with 1 ‰ Tween ice for 5min each time in a shaker. One wash with ice PBS for 5min on a shaker. Preparing a fluorescence-labeled secondary antibody: PE-labeled Goat anti-rabbit immunoglobulin G (Goat anti-rabbit IgG (H & L) TRITC, Abcam, USA, Abcam 50598) in PBS or FBS at a concentration of 1: 200. Secondary antibody was added and incubated for 1 hour at room temperature (protected from light). Washed twice with 1 ‰ Tween ice for 5min each time in a shaker. One wash with ice PBS for 5min on a shaker. DAPI stained nuclei, 1 drop per dish, completely covering the cells. Washed twice with 1 ‰ Tween ice for 5min each time in a shaker. One wash with ice PBS for 5min on a shaker. Adding the anti-fluorescence quenching sealing tablet and keeping out light. Observing and taking a picture by using a machine confocal immunofluorescence microscope.

FIG. 4 is an immunofluorescence chart of the differentiation of OPCs transfected by 10 th generation recombinant GAP43/GGF2 lentivirus into oligodendrocytes. The figure is an oligodendrocyte immunofluorescence image observed by a 4-fold objective lens, wherein green star-shaped GFAP stained oligodendrocytes can be observed, and the result shows that 10 th generation recombinant GAP43/GGF2 lentivirus transfected OPCs can be differentiated into oligodendrocytes.

Example 6 in vivo experiments in transplantation of rat model for spinal cord injury GAP43/GGF2-OPCs

20 experimental SD rats purchased from the center of animals of military medical academy of sciences, 3% pentobarbital sodium (30mg/kg body weight) is used for carrying out intraperitoneal injection and anesthesia on the rats, the rats are fixed on an operating table in a prone position, depilation, disinfection and towel paving are carried out, T12 is positioned at the highest position of the back of the rats, the rats are cut open in a central posterior longitudinal shape, the rats grow about 1.5cm in length, paraspinal muscles are retracted to two sides, T12 spinous processes and vertebral laminae are exposed, the T12 spinous processes and the vertebral laminae are bitten off to form a bone window with the rear diameter of 4.0mm, the vertebral canal and the dura are fully exposed and reach the spinal dura mater spinalis, a round metal impact bar with the weight of 10.0g (the lower end of the metal impact bar has the diameter of about 2.5mm and is almost matched with the diameter of the spinal cord) is used for passing through a hollow tube with the inner diameter of 4.0mm and the height of 7cm away from the spinal cord, the hollow tube, and detecting body-induced evoked potentials after operation, further evaluating the credibility of the spinal cord impact test, and rejecting the animals which fail impact.

The established rat model of spinal cord injury was randomly divided into A, B and C groups of 6 animals each, wherein group A was the transplanted group of OPCs transfected by recombinant GAP43/GGF2 lentivirus prepared in example 3, group B was the transplanted group of OPCs prepared in example 2, and group C was the transplanted group of physiological saline.

After the rat model with spinal cord injury is raised in cages for 72 hours, group A is transplanted with tail vein GAP43/GGF2-OPCs 2 × 10⁶100 μ l/kg, group B for tail vein transplantation OPCs 2 × 10⁶100 mul/kg; group C was given tail vein injections of 100. mu.l of physiological saline; the results of Basso Beattie Bresnahan (BBB) motor function scoring on all rats on the day of transplantation, 1d, 3d, 5d, 7d, 14d, 28d, are shown in Table 1 below.

TABLE 1

Description of the drawings: BBB motor function score of 0-7 judges the movement of each joint of hind limb of animal; 8-13, judging the gait and coordination function of hind limbs; the fine movements of the paws in the sports are judged in 14-21 points, the three items are divided into 21 points, and the higher the score is, the smaller the damage is.

As can be seen from Table 1, p values and^#p values were all much less than 0.05, and p values were much less than 0.05 in group a compared to group B starting 14 days after transplantation; BBB rating of groups A and BThe composition is divided into a group A and a group C, wherein the BBB score of the group A is obviously higher than that of the group B, namely the OPCs transfected by GAP43/GGF2 lentivirus of the invention used for transplanting spinal cord injury have excellent treatment effect and small immunological rejection, and the OPCs transfected by GAP43/GGF2 lentivirus and untransfected OPCs promote the nerve function recovery of spinal cord injury rats, wherein the OPCs transfected by GAP43/GGF2 lentivirus have more obvious effect.

Example 7 spinal cord injury rat model GAP43/GGF2-OPCs transplanted cortical somatosensory evoked potential analysis

After the OPCs transfected by the recombinant GAP43/GGF2 lentivirus prepared in example 3 and the OPCs prepared in example 2 are transplanted, a rat model with spinal cord injury prepared in example 5 is used, an evoked potential instrument (Shanghai Shen medical electronics Co., Ltd.) is used for detection, a stimulation electrode is arranged in the middle of the tibialis anterior muscle, a reference electrode is arranged at the far side of 1cm, the stimulation intensity is 1.5-4 mA, the wave width is 0.2mA, and the frequency is 1.9Hz and continuous square wave stimulation is superposed for 20-40 times. The recording electrode is placed under the scalp at the intersection of the midline of the top of the head and the coronal suture, and the reference electrode is placed 0.5cm behind the recording electrode. The ground wire is clung to the tail of the animal soaked by the normal saline. Changes of Cortical Somatosensory Evoked Potentials (CSEP) P1-N1 wave amplitudes before injury, on the day of transplantation, 14 days after transplantation and 28 days after transplantation of the rats are respectively observed based on slight twitching of hind limbs of the rats.

TABLE 2

From table 2, it can be seen that CSEP amplitude was significantly reduced on the day after spinal cord injury in rats. At 14d post-transplantation, the amplitude was significantly increased in group a, with statistical differences compared to groups B and C (. p < 0.05). After transplantation, the CSEP wave amplitude of the group C is obviously increased at 28d, and the difference is statistically significant compared with that at 14d (& p is less than 0.05); meanwhile, the CSEP wave amplitude of the group A is obviously increased when being compared with that of the group C, the difference has statistical significance (P is less than 0.05), and compared with the group B and the group C at the same period, the difference also has statistical significance (P is less than 0.05); furthermore, the amplitudes of 14d and 28d CSEP after the B group transplantation are obviously increased, and the B group CSEP also has statistical significance compared with the C group at the same period (# p < 0.05). The results show that the GAP43/GGF2-OPCs transplanted spinal cord injured rat has the function of recovering the nerve function and is obviously better than the simple OPCs.

Example 8 spinal cord injury rat model GAP43/GGF2-OPCs transplanted glial scar assay

Example 5 spinal cord injury rat model on day 28 after OPCs transplantation, 1cm spinal cord tissue was removed immediately after cervical amputation and fixed in 4% paraformaldehyde for 48 hours. Paraffin embedding, spinal cord tissue section (5 μm), immunohistochemistry was performed to detect the expression level of GFAP. Paraffin sections were attached to polylysine-treated slides and allowed to stand at room temperature for 60 minutes prior to deparaffinization. Sequentially slicing the paraffin into dimethylbenzene I for 10 minutes; xylene II, 10 minutes; absolute ethyl alcohol, 5 minutes; 95% ethanol, five minutes; 75% ethanol, five minutes; 50% ethanol, five minutes; deionized water, five minutes. PBS was washed 3 times for 5 minutes each. The samples were digested with 0.1% pancreatin for 30 min at 37 ℃ in a wet box for antigen retrieval; removing the enzyme solution, washing with PBS for 5 minutes for 3 times; endogenous peroxidase was inactivated by treatment with 3% H2O2 deionized water for 10 minutes. PBS was washed 3 times for 5 minutes, 50. mu.l of primary anti-rabbit anti-mouse GFAP (1: 500) (Millipore, USA) was added dropwise, and after overnight at 4 ℃, PBS was washed 3 times for 5 minutes each; adding 50 μ l of alkaline phosphatase-labeled anti-mouse secondary antibody (1: 1000) (purchased from Beijing Zhonghua company), and standing at 37 deg.C for 1 hr; washing with PBS for 5 minutes for 3 times, dripping fresh DAB for developing for 10 minutes, and grasping the dyeing degree under a microscope; washing with PBS for 10 min; counterstaining with hematoxylin for 2 minutes, and differentiating with hydrochloric acid and alcohol; washing for 10 minutes by tap water; dehydrating, sealing with transparent neutral resin, and taking pictures by microscope observation.

From fig. 5, we can see that the damaged segment of the spinal cord injury part in the group C28 d has more expression quantity with GFAP positive cells in the groups A and B, the positive cells have larger cell bodies and thicker and longer protrusions, and the damaged segment and the GFAP positive cells are mutually tangled and interwoven into a net shape to form a compact colloid scar; when the expression quantity of the GFAP positive cells in the group A is 28 days, the GFAP positive cells are obviously reduced compared with the group B and the group C, the volume of the GFAP positive cells is reduced, the protrusions are reduced, the staining is lightened, the range of glial scar is reduced, and the fact that GAP43/GGF2-OPCs are transplanted to treat spinal cord injury rats to inhibit astrocyte proliferation and glial scar formation is shown.

Claims

1. A method for preparing human Oligodendrocyte Precursor Cells (OPCs) for inhibiting nerve secondary injury comprises constructing virus expression vectors of two glial growth factors, packaging into viruses, co-transfecting the human oligodendrocyte precursor cells, and preparing the oligodendrocyte precursor cells which can highly express the two glial growth factors and have no tumorigenic activity; wherein the glial growth factor is growth-related factor 43(GAP-43) and glial growth factor 2 (GGF-2);

in a rat model with spinal cord injury, the motor function score improvement value of BBB (Basso Beattie Bresnahan) 28 days after the transplantation of OPCs cotransfected by GAP43/GGF2 virus is 20.00 +/-5.00, and the amplitude improvement value of cortical somatosensory evoked potential CSEP is 6.00 +/-1.00;

GAP43/GGF2-OPCs inhibit astrocyte proliferation and glial scarring in the treatment of spinal cord injured rats.

2. The method of claim 1, wherein the growth-related factor 43 and glial growth factor 2cDNA sequences are amplified by polymerase chain reaction technology, and are recombined onto expression plasmids respectively, and are connected to viral expression vectors through enzyme cleavage sites, and the two genetically recombinant viral vectors co-transfect human oligodendrocyte progenitor cells to obtain human oligodendrocyte progenitor cells with enhanced proliferative capacity and differentiation capacity.

3. The method of claim 2, wherein the oligodendrocyte precursor cells are derived from in vitro culture of human waste cord blood, placental blood, umbilical cord, or autologous bone marrow.

4. The method according to any one of claims 1 to 3, wherein the oligodendrocyte precursor cells are prepared by taking mononuclear cells isolated and purified from 20mL of bone marrow by lymphocyte separation medium, and resuspending the mononuclear cells in serum-free oligodendrocyte medium to 2-5 × 10⁶Per mL, pipette 10mL into 10-200. mu.g/mL polylysine coated 75cm²In a cell culture flask, 5% CO at 37 ℃₂Culturing in a cell culture box for 48 hours; replacing a fresh serum-free oligodendrocyte medium containing the cell factors, continuously culturing until the growth and fusion of the cells reach 90%, digesting by using pancreatin with the mass-to-volume ratio of 0.25%, carrying out subculture, namely 1 bottle of subculture and 2 bottles of subculture, supplementing the fresh serum-free oligodendrocyte medium containing the cell factors, and continuously culturing to obtain the human oligodendrocyte progenitor cells; wherein the cell culture medium is DMEM/F12 containing 1% N2, 2mM L-glutamine, 5-100ng/mL basic fibroblast growth factor, 1-50ng/mL fibroblast growth factor 4, 1-100ng/mL stem cell growth factor, and 1-100ng/mL FMS-like tyrosine kinase 3 ligand; the pancreatin contains EDTA with the mass volume ratio of 0.02 percent; the human oligodendrocyte precursor cells are detected by a flow cytometry technology, and OPCs markers A285, O4 and Sox10 are all expressed by more than 95%.

5. A method for preparing human oligodendrocyte precursor cells for inhibiting nerve secondary injury,

amplifying human GAP-43 and GGF-2 sequences from normal human peripheral blood genome DNA by PCR, respectively carrying out ligation reaction on GAP-43 and GGF-2 target fragments and an XhoI/Nde I double-enzyme digested pET28a vector fragment at 4 ℃ for 12 hours under the action of T4DNA ligase to prepare a clone ligation solution, and carrying out positive clone PCR identification and sequencing identification after transforming an escherichia coli competent cell DH5 alpha; after PCR product gel electrophoresis detection and sequencing identification accord with the size and sequence of GAP-43 and GGF-2, transferring the correctly sequenced bacterium liquid into 10mL LB liquid culture medium containing corresponding antibiotics, culturing overnight at 37 ℃, performing plasmid extraction by using an endotoxin-free plasmid small-extraction medium-amount kit, and placing the qualified recombinant plasmid in an ultra-low temperature refrigerator at-80 ℃ for long-term storage; the sizes of the GAP-43 gene fragment and the GGF-2 gene fragment are 706bp and 1268bp respectively;

carrying out XhoI/Nde I double enzyme digestion on GAP-43, GGF-2DNA fragments and GV358 vectors respectively, carrying out ligation reaction on the GAP-43 fragments, the GV358 vectors, the GGF-2 fragments and the GV358 vectors at 37 ℃ for 12 hours respectively under the action of T4DNA ligase to prepare clone ligation liquid, and carrying out positive clone PCR identification and sequencing identification after transforming escherichia coli competent cells DH5 alpha;

293T cells in a good cell state and in a logarithmic growth phase were collected, and after counting, the cells were cultured in 10cm dishes 6 × 10⁶The number of cells was plated in a petri dish at 37 ℃ with 5% CO₂Culturing in an incubator overnight; removing the culture solution before transfection on the next day, and replacing 5mL of Opti-MEM culture solution; adding 9 μ g of the packaged mixed solution and 3 μ g of the lentivirus expression plasmid into 1.5mL of Opti-MEM, gently mixing, adding 36 μ l of lipofectamine2000 into 1.5mL of Opti-MEM, gently mixing, and standing at room temperature for 5 min; mixing the plasmid solution and lipofectamine2000 diluent, and standing at room temperature for 20 min; the mixed solution is slowly dripped into the 293T cell culture solution, mixed evenly and treated at 37 ℃ with 5% CO₂Culturing in a cell culture box; culturing for 6h, discarding the culture medium containing the transfection mixture, adding 10mL of PBS (phosphate buffer solution) for washing once, gently shaking the culture dish to wash the residual transfection mixture, and then pouring and discarding; slowly adding 20mL of 10% serum-containing cell culture medium, and adding 5% CO at 37 deg.C₂Continuously culturing for 48-72h in the incubator;

collecting 293T cell supernatant 48h after transfection according to cell state; centrifuging at 4000g for 10min at 4 deg.C to remove cell debris; the supernatant was filtered through a 0.45 μm filter into a 40mL ultracentrifuge tube; respectively balancing samples, putting ultracentrifuge tubes with virus supernatant into a Beckman ultracentrifuge one by one, setting the centrifugation parameters to be 25000rpm, setting the centrifugation time to be 2h, and controlling the centrifugation temperature to be 4 ℃; after the centrifugation is finished, removing the supernatant, removing the liquid remained on the tube wall as much as possible, adding a virus preservation solution, and lightly and repeatedly blowing and resuspending; after full dissolution, centrifuging at high speed of 10000rpm for 5min, taking supernatant to measure titer by a fluorescence method, subpackaging the packaged GAP-43 and GGF-2 lentivirus according to 50 mu l of 2E +8TU/mL, and storing in an ultra-low temperature refrigerator at-80 ℃;

adding 10 μ l of each of the recombinant GAP-43 and GGF-2 lentivirus 1E +7TU/mL into a 10-200 μ g/mL polylysine-coated six-well culture plate of human oligodendrocyte progenitor cells in a system of 2mL, mixing, 37 deg.C, and 5% CO₂After 8-12 hours of incubation in the incubator of (a), complete medium DMEM/F12 was replaced, said complete medium DMEM/F12 containing 1% N2, 2mM L-glutamine, 5-100ng/mL basic fibroblast growth factor, 1-50ng/mL fibroblast growth factor 4, 1-100ng/mL stem cell growth factor and 1-100ng/mL FMS-like tyrosine kinase 3 ligand; when the cell growth fusion reaches 90%, the cells are digested with 0.25% pancreatin and transferred to 25cm coated with 10-200. mu.g/mL polylysine²Growth in flasks, 1 well for 1 flask at 25cm²Continuing digestion subculture when the fusion in the culture flask reaches 90%; the growth transfections were divided into 3 groups: untransfected control group, blank lentivirus transfection group, co-transfected GAP-43 and GGF-2 lentivirus transfection group;

observing the luminescence condition of Green Fluorescent Protein (GFP) under a fluorescence microscope 72 hours after infection, wherein the GFP color development reaches over 90 percent, which indicates that GAP-43 and GGF-2 lentivirus are transfected successfully to transfect human oligodendrocyte progenitor cells, and the human oligodendrocyte progenitor cells which continuously express GAP-43 and GGF-2 are obtained;

detecting by a fluorescent quantitative PCR kit according to an instruction, after the transfected GAP-43 and GGF-2 lentivirus transfected human oligodendrocyte progenitor cells are subcultured to the 10 th generation, the expression GAP-43 and GGF-2 genes of the cells are respectively higher than 45.89 times and 67.98 times of untransfected OPCss;

the method of producing the oligodendrocyte precursor cells according to claim 4.

6. The production method according to any one of claims 1 to 3 and 5,

OPCs transfected with GAP-43 and GGF-2 lentiviral vectors are subjected to in-vitro subculture by complete culture solution DMEM/F12, when the cells are cultured for 5-6 days, the cells grow and fuse to more than 90%, and pancreatin with the mass-volume ratio of 0.25% is used for digestion and subculture;

taking the cell morphology observed under an inverted microscope of OPCs cultured to 3 generations and 10 generations, the untransfected OPCs and other OPCs transfected with target genes have similar astrocyte morphology, and oligodendrocyte can be differentiated.

7. The method according to any one of claims 1-3 and 5, wherein the human oligodendrocyte precursor cells highly express growth-related factor 43 and glial growth factor 2 and have stem cell morphology when observed under an inverted microscope after subculture to 10 passages; the multiplication times of the cells reach more than 500 times after subculture until the 10 th generation.

8. A method for differentiating human oligodendrocyte progenitor cells transfected by recombinant GAP43/GGF2 lentivirus into oligodendrocytes is characterized in that,

inoculating 10 th generation of OPCs transfected by recombinant GAP43/GGF2 lentivirus to a 6-hole culture plate previously provided with a polylysine coating cover glass with the concentration of 50 mu g/mL, inducing liquid DMEM/F12 after the cells grow to be completely fused, and culturing at 37 ℃ and 5% CO₂Culturing in incubator for 24 hr, removing inducing solution, adding completely differentiated culture solution DMEM/F12, and culturing at 37 deg.C under 5% CO₂Culturing in an incubator, changing the culture solution 1 time every 72h, and culturing for 7 days; after 7 days of culture, the cells were fixed with 4% paraformaldehyde, and subjected to Glial Fibrillary Acidic Protein (GFAP) immunocytochemistry;

GAP43/GGF2-OPCs are fixed by 4% cold paraformaldehyde for 15 minutes and then are protected from light; after absorbing paraformaldehyde, washing with ice PBS for three times, 5 minutes each time; 0.5% Triton X-100 covered the cells for 10min, washed three times with ice PBS, 5min each; sealing the imported fetal goat serum of the same host as the secondary antibody for 30 minutes at room temperature; preparing a primary antibody: diluting rabbit anti-human GFAP (serum-derived serum) 200 times with fetal sheep serum, adding primary antibody covering cells, and wrapping with tinfoil at 4 ℃ in a dark place overnight; the next day: taking out the cells and rewarming the cells to room temperature for about 1 h; washing twice with ice 1 ‰ Tween for 5min in shaking bed; washed once with ice PBS for 5 minutes on a shaker; preparing a fluorescence-labeled secondary antibody: PE-labeled goat anti-rabbit immunoglobulin G prepared with PBS or FBS at a concentration of 1: 200; adding a secondary antibody, incubating for 1 hour at room temperature, and keeping out of the sun; washing twice with ice 1 ‰ Tween for 5min in shaking bed; washed once with ice PBS for 5 minutes on a shaker; staining nuclei with DAPI, wherein 1 drop of the DNA stain is applied to each dish and completely covers cells; washing twice with ice 1 ‰ Tween for 5min in shaking bed; washed once with ice PBS for 5 minutes on a shaker; adding the anti-fluorescence quenching sealing tablet and keeping out of the sun; observing and taking a picture by using an on-machine confocal immunofluorescence microscope; the results showed that GFAP-stained oligodendrocytes were observed as green stars, indicating that generation 10 recombinant GAP43/GGF2 lentiviral-transfected OPCs could differentiate into oligodendrocytes;

wherein the GAP43/GGF2-OPCs are prepared by the method of any one of claims 1-7.

9. A kit for preparing human oligodendrocyte precursor cells for inhibiting secondary damage to nerves prepared by the method of any one of claims 1-7, wherein the kit comprises:

1) basal medium for human oligodendrocyte progenitor cells;

3) an enzyme that digests the cells;

4) a cytokine and;

5) instructions for use;

wherein the instructions for use comprise the method of any one of claims 1-7.

10. A human oligodendrocyte precursor cells for inhibiting secondary damage to a nerve prepared by the method of any one of claims 1-7.