CN113583965A - Condition immortalized human neural stem cell-derived cell membrane nano vesicle preparation as well as preparation method and application thereof - Google Patents
Condition immortalized human neural stem cell-derived cell membrane nano vesicle preparation as well as preparation method and application thereof Download PDFInfo
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- CN113583965A CN113583965A CN202110898232.3A CN202110898232A CN113583965A CN 113583965 A CN113583965 A CN 113583965A CN 202110898232 A CN202110898232 A CN 202110898232A CN 113583965 A CN113583965 A CN 113583965A
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- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/82—Translation products from oncogenes
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/999—Small molecules not provided for elsewhere
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2510/00—Genetically modified cells
- C12N2510/04—Immortalised cells
Abstract
The invention discloses a conditionally-immortalized human neural stem cell-derived cell membrane nano vesicle preparation as well as a preparation method and application thereof, belonging to the technical field of cell engineering and genetic engineering. The human neural stem cells are transformed by a genetic engineering technology to become conditionally-immortalized cells, and can be amplified in vitro in large quantities. Then, the physical extrusion technology is used for promoting the cell membranes of conditionally-immortalized human neural stem cells to be fused again, so that the cell membrane nano vesicles with the characteristics of parent cells are prepared in a large scale, a set of complete system from cells to subsequent preparation of cell derivatives is constructed, and the problems that the preparation of the human neural stem cell derivatives facing clinical transformation is difficult to produce in a large scale and the like can be solved. The invention provides a powerful foundation for applying the human neural stem cell derivative preparation to clinic by establishing a preparation method of the conditionally immortalized human neural stem cell derived cell membrane nano vesicle preparation.
Description
Technical Field
The invention belongs to the technical field of cell engineering and genetic engineering, and particularly relates to a conditionally-immortalized human neural stem cell-derived cell membrane nano vesicle preparation as well as a preparation method and application thereof.
Background
Development of CNS-related therapeutics has met with limited success due to the inherent complexity of the Central Nervous System (CNS). In recent years, stem cell therapy has promoted the clinical progress of a variety of intractable central nervous system diseases and achieved certain therapeutic effects worldwide. Particularly, neural stem cells have a more effective role in the treatment of nervous system diseases as seed cells derived from the central nervous system. A large number of preclinical studies indicate that neural stem cell transplantation as a potential therapeutic approach plays an important role in central nervous system diseases through various mechanisms such as neurotrophic factor generation, neuroinflammation reduction, neural plasticity enhancement, cell replacement and the like. However, to date, only a few clinical studies have been published, and clinical application of neural stem cells has presented unprecedented challenges. The main obstacles are the difficulty of highly complex life processes to be controlled by in vitro processes, the low survival of transplanted cells, the risk of immune rejection and tumor formation, and the concern of whether these cells are suitable for further use. In addition, the properties of neural stem cells make transplantation more difficult, including low proliferation rates, limited sources, ethical issues, and the like. Therefore, there is a need to develop an effective carrier with intrinsic bioactivity that retains function and activity while reducing the risks associated with stem cell therapy, such as unwanted replication, differentiation and vascular occlusion.
Neural stem cell-derived extracellular vesicles (Evs) provide a particularly attractive alternative to cell transplantation for basal therapies, and unlike cellular therapies, the probability of safety problems following Evs administration is almost zero because they do not have nucleated cells, cannot replicate, and degrade quickly upon entry into the cells. Many studies have shown that neural stem cell-mediated functional recovery is primarily due to trophic support provided by neural stem cell paracrine signals, including growth factors and extracellular vesicles, such as exosomes. Stem cells are used as producers of therapeutic drugs, rather than as therapeutic drugs themselves, which enhances the feasibility of therapy. Therefore, cell-free therapy has attracted great interest. In order to enable cell-free products to replace cell therapy as safer, cheaper biopharmaceuticals to enter the clinic, the large-scale production of cell-free therapies is a major challenge for manufacturing. Therefore, it is highly desired to develop a neural stem cell-derived cell derivative preparation which is highly efficient and can be produced on a large scale.
Disclosure of Invention
In view of the above, the present invention aims to provide a cell membrane nanovesicle preparation derived from conditionally-immortalized human neural stem cells and a preparation method thereof, which construct a set of complete system from cell preparation to subsequent cell derivative preparation, and is used for solving the problem that the large-scale production of human neural stem cell derivatives is difficult to achieve in clinical transformation.
The purpose of the invention is realized by the following modes:
the invention firstly transforms the human neural stem cells by the genetic engineering technology, constructs the conditionally immortalized human neural stem cells, accelerates the proliferation rate of the conditionally immortalized human neural stem cells, realizes large-scale culture of the conditionally immortalized human neural stem cells, and ensures the stability of the preparation source of the cell membrane nano vesicle preparation.
A preparation method of a conditionally-immortalized human neural stem cell-derived cell membrane nano vesicle preparation mainly comprises the following steps:
(1) constructing conditionally-immortalized human neural stem cells by a genetic engineering means, wherein the conditionally-immortalized human neural stem cells have an increased proliferation speed under the action of a specific medicament and can be subjected to large-scale amplification culture;
(2) adding the specific medicine into the culture system of the conditionally-immortalized human neural stem cells obtained in the step (1), improving the proliferation speed of the conditionally-immortalized human neural stem cells, and obtaining a large batch of conditionally-immortalized human neural stem cells after culture;
(3) promoting the conditionally-immortalized human neural stem cell membranes obtained in the step (2) to be fused again by a physical extrusion method, and obtaining the cell membrane nano vesicles with maternal cell characteristics.
Based on the technical scheme, further, the immortalized human neural stem cell expresses a c-myc ER system (estrogen-induced c-myc system) in the step (1).
Based on the technical scheme, the c-myc ER system is further formed by fusing c-myc and an estrogen receptor ER.
Based on the technical scheme, further, the nucleotide sequence of the c-myc ER system is shown as SEQ ID NO. 1.
Based on the technical scheme, the specific process for constructing the conditionally-immortalized human neural stem cell in the step (1) is to directly introduce a gene encoding the c-myc ER system into a host cell or to introduce a recombinant virus carrying the gene encoding the c-myc ER system into the host cell by a virus transfection mode.
Based on the above technical scheme, further, the ligand binding region of estrogen receptor of c-myc ER system expressed by the conditionally-immortalized human neural stem cells comprises point mutation (G521R).
Based on the technical scheme, further, the specific medicine comprises tamoxifen and 4-hydroxy tamoxifen.
Based on the technical scheme, further, the final concentration of the specific drug in the culture system is 10-1000 nM.
Based on the technical scheme, the specific drug has reversibility to regulation and control of the proliferation rate of the immortalized cells, when the drug is added into the immortalized cells, the reactive proliferation rate of the immortalized cells is accelerated, and when the drug addition is stopped, the proliferation rate of the immortalized cells can be gradually recovered to the original level.
Based on the technical scheme, further, the specific operation steps of the physical extrusion method in the step (3) include passing the cell suspension of the conditionally immortalized human neural stem cells through a filter membrane with the pore size of 10 μm, 5 μm and 1 μm in sequence by an extruder, repeatedly extruding for 5-20 times, collecting the obtained suspension, centrifuging for 5-30 min at 0-4 ℃ at 1000-5000 g, taking the supernatant, centrifuging for 5-30 min at 0-4 ℃ at 10000-30000 g, discarding the supernatant, and resuspending the obtained precipitate with a buffer solution to obtain the cell membrane nanovesicles.
The invention also provides a cell membrane nano vesicle, which is prepared by the preparation method.
The invention also provides application of the cell membrane nano-vesicle in preparation of a medicament for treating central nervous system diseases.
Compared with the prior art, the invention has the following beneficial effects:
1. the invention discloses a condition immortalized human neural stem cell source cell membrane nano vesicle preparation capable of being prepared in a large scale and a preparation method thereof. Then preparing the cell membrane nano vesicles which have the characteristics of parent cells and are derived from conditionally-immortalized human neural stem cells on a large scale by a cell membrane re-fusion promoting technology through physical extrusion. A set of complete system from cell to subsequent preparation of cell derivative preparation is constructed, and the problem that the preparation is difficult to produce on a large scale when the human neural stem cell derivative preparation faces clinical transformation can be solved.
2. The preparation method of the conditionally-immortalized human neural stem cell membrane nanovesicle preparation capable of being prepared in a large scale has the characteristics of high yield, simplicity in operation, strong repeatability and the like. Meanwhile, the selected condition immortalization construction scheme can regulate and control the proliferation rate of the human neural stem cells by adding medicaments from an external source, thereby ensuring the safety of the maternal cells from preparations.
3. The invention provides a whole set of complete scheme from cell to cell derivative preparation by establishing a preparation method of the cell membrane nano vesicle preparation derived from the immortalized human neural stem cell under the condition, and provides reference for the clinical transformation research of the subsequent cell derivative preparations derived from other cells.
Drawings
In order to more clearly illustrate the embodiments of the present invention, the drawings to which the embodiments relate will be briefly described below.
FIG. 1 is a flow chart of the preparation method of the conditionally-immortalized human neural stem cell-derived cell membrane nanovesicle preparation of the present invention;
FIG. 2 is an optical microscope photograph of human neural stem cells of different generations ( generations 2, 4 and 8) in example 1 of the present invention;
FIG. 3 is a picture showing the identification of specific markers for human neural stem cells in example 1 of the present invention;
FIG. 4 is a photograph showing the measurement of the differentiation inducing ability of human neural stem cells in example 1 of the present invention;
FIG. 5 is a map of a lentiviral expression vector encoding an estrogen-inducible c-myc system, as described in example 1 of the present invention;
FIG. 6 is a photograph under a fluorescent microscope of human neural stem cells after infection with lentivirus in example 1 of the present invention;
FIG. 7 is a photograph showing karyotype analysis of conditionally-immortalized human neural stem cells according to the present invention;
FIG. 8 is a photograph showing the transcription of downstream genes of the recombinant human neural stem cells in example 1 of the present invention after adding 4-hydroxyttamoxifen, wherein 4-OHT is the drug-adding group and un-4-OHT is the complete medium experimental group;
FIG. 9 is a graph showing the change in proliferation rate of the recombinant human neural stem cells in example 1 of the present invention without or with the addition of 4-hydroxytamoxifen, wherein 4-OHT is the drug-added group and un-4-OHT is the complete medium experimental group;
FIG. 10 shows the human neural stem cells and recombinant human neural stem cells EdU labeling detection proliferation assay of the present invention;
FIG. 11 is a Western Blot of conditional immortalized human neural stem cell-derived cell membrane nanovesicles of example 6 according to the present invention;
FIG. 12 is a transmission electron microscope image of nanovesicles of cell membranes derived from conditional immortalized human neural stem cells according to example 6 of the present invention;
FIG. 13 is a graph showing the distribution of the diameters of nanovesicles in cell membranes derived from the condition-immortalized human neural stem cells in example 6 of the present invention;
FIG. 14 is the construction of a model of hypoxic injury to neurons in example 7 of the present invention;
FIG. 15 shows apoptosis of immortalized human neural stem cell-derived cell membrane nanovesicles after treatment in example 7 of the present invention.
Detailed Description
The present invention is described in detail below with reference to examples, but the embodiments of the present invention are not limited thereto, and it is obvious that the examples in the following description are only some examples of the present invention, and it is obvious for those skilled in the art to obtain other similar examples without inventive exercise and falling into the scope of the present invention.
In the following examples, unless otherwise specified, the experimental methods used were all conventional methods, and materials, reagents and the like used were all available from biological or chemical reagents companies.
Example 1: culture and identification of human neural stem cells
The invention improves the proliferation capacity of human Neural Stem Cells (NSCs) by modifying the NSCs. First, human neural stem cells are cultured and identified.
Human neural Stem cells were cultured in suspension in neural Stem cell complete medium (purchased from Stem cell technology, catalog # 05751). The specific operation mode is as follows:
the primary neural stem cell extraction preparation is carried out in a biological safety cabinet, and strict aseptic operation is not required in the operation process. Taking a sterile culture dish with the diameter of 100mm, pouring 10mL of sterile normal saline, and repeatedly washing the complete aborted fetal tissues (which accord with the ethical guidelines of human embryonic stem cell research) in the normal saline solution until the normal saline is clear and transparent. Use of sterilized surgical scissors and forceps to strip the skin of the head of an aborted fetusSkin, cut the skull to fully expose the brain tissue, gently tear off the pia mater around the brain tissue with the surgical forceps, and peel off the blood vessel as much as possible. Taking out the fetal brain tissue completely, placing into a new culture dish containing sterile normal saline, and carefully separating the cerebral cortex. A35 mm sterile petri dish was prepared, a neural stem completion medium was placed in the dish, and the separated cortical tissue was placed in the medium and divided into tissue pieces of 1mm in size. Transferring the complete culture medium containing the tissue block into a 50mL centrifuge tube by using a pipette, gently blowing and beating for 30 times, putting into an incubator at 37 ℃, standing for 10 minutes, taking the supernatant, putting into a new 50mL centrifuge tube, centrifuging for 5 minutes at 300g, and discarding the supernatant. 2mL of Accutase (purchased in Stem cell technology, catalog #07920) was added to a 50mL centrifuge tube, gently mixed, and the tube was placed at 37 ℃ in a 5% CO2The incubator was digested for 1min, centrifuged at 300g for 5min at room temperature, and the supernatant was discarded. Add 1mL NSC complete medium to the centrifuge tube, gently blow and beat until a single cell suspension is formed and count the cells. Taking a T75 cell culture bottle, adding 20mL of complete medium, and counting the cells at 2x104～5x104The cells were inoculated into a culture flask at a density of one/mL, and the cell batch number, the cell generation number, the name of the operator, and the operation time were marked. After inoculation, the cells are placed under a common optical microscope for microscopic observation, the cells are approximately uniformly distributed in each culture container, and if the cells are not uniformly distributed, the containers are shaken again. Cellular status and abnormalities were recorded. Placing the cells after microscopic examination into a carbon dioxide incubator at 37 deg.C and CO2The concentration was 5%. And 2-3 days after cell inoculation, observing under a microscope, recording the growth state of the cells, and performing fluid infusion, wherein the volume of the fluid infusion is 2-3 mL. After that, cell exchange is carried out every 3 days, suspension culture of primary cells is carried out after extraction, and neural stem cell passage is carried out when the neurosphere grows to 100-150 mu m.
Transferring the culture solution containing the neurospheres into a 15 or 50mL centrifuge tube, centrifuging at room temperature for 2 minutes at 200g, discarding most of supernatant after centrifugation to enable the neurospheres to be left in a small volume of culture medium, adding 1mL of Accutase enzyme, incubating at 37 ℃ for 5 minutes, and intermittently and slightly blowing to ensure that the cells do not aggregate or precipitate at the bottom of the test tube. The neurosphere is gently blown and beaten by using a 1000 mu L gun headA single cell suspension was formed and digestion was stopped by adding 4mL of complete medium. Centrifuging at room temperature for 3min at 300g, discarding the supernatant, resuspending the cells in fresh medium, and diluting the cells to 2-5 x104Inoculating into culture flask at density of one/mL, placing at 37 deg.C, and adding 5% CO2The incubator is continued to be cultured.
The cell morphology of the human neural stem cells of different generations is observed through an optical microscope, and the result is shown in fig. 2, so that the growth state of the cells of different generations is good, no obvious difference exists between different generations, and the stability of the extracted human neural stem cells in-vitro culture is further proved.
Based on human neural stem cell high-expression Nestin and Sox2, the human neural stem cell is identified by a flow cytometer with a specific marker, and the specific operation steps are as follows: digesting cells by using Accutase enzyme, adding a complete neural stem cell culture medium to blow the neural stem cells to single cells, performing membrane rupture and fixation on the obtained neural stem cells for 15min by using 4% paraformaldehyde, centrifuging for 5min at 300g, removing the supernatant, adding 2mL of 1 XPBS, fully mixing, centrifuging for 5min at 300g again, removing the supernatant, adding 1 XPBS for resuspension, and preparing a single cell suspension (the cell density is 1 XP 10)6/mL). 100ul of the suspension was added to a labeled flow tube, and incubated with the desired antibody (Nestin antibody, Sox2 antibody) for 30min in the absence of light. After incubation, 1ml of 1 XPBS is added into each tube, the mixture is fully mixed, 300g of the mixture is centrifuged for 5 minutes, supernatant is discarded, and after washing twice, the mixture is put on a computer to select the corresponding wavelength of the corresponding antibody for detection.
The identification result of the human neural stem cell specific marker is shown in figure 3, and the result shows that the human neural stem cell cultured by the invention has high expression of Nestin and Sox2, has no differentiation, and is a normal neural stem cell before establishing a lineage.
The human neural stem cells of the present invention were examined for their ability to differentiate based on their ability to differentiate into neurons, astrocytes and oligodendrocytes. The specific operation steps are as follows: the neural stem cells are arranged according to the 4 x105Culturing the cells/well in matrigel-coated six-well plate, and proliferating the neural stem cell culture mediumNeural Stem cell differentiation medium (purchased from Stem cell technology, #05752) was replaced by 2ml per well. Changing a differentiation medium every 2-3 days, carrying out immunofluorescence staining identification on a CQ1 laser confocal high-content cell screening system (YOKOGAWA, CQ1) after differentiation induction culture for 10 days, and verifying that the extracted human neural stem cells have the capacity of differentiating into three nerve cells of a nervous system.
The results of the measurement of the induced differentiation capacity of human neural stem cells are shown in FIG. 4, in which A: cell morphology observed under a common optical microscope on the 2 nd day and the 4 th day of induced differentiation of the neural stem cells; b: and (3) carrying out immunofluorescence staining on neurons: mouse monoclonal antibody beta III Tubulin (Tuj1) and DAPI staining; c: oligodendrocyte immunofluorescent staining: staining rabbit polyclonal antibody Oligodendrocyte Specific Protein and DAPI; d: immunofluorescence staining of astrocytes: staining rabbit polyclonal antibodies GFAP and DAPI; further proves that the human neural stem cells extracted by the invention have the capacity of differentiating into three nerve cells of the nervous system.
Example 2: construction of expression vectors
This example modifies cells by a method of lentivirus infection of host cells. Therefore, firstly, an expression vector containing an estrogen-inducible c-myc system is constructed, a c-myc ER gene is constructed in a lentiviral vector, and the used lentiviral expression vector is GV492 (purchased from Kjekay Gene chemistry, Inc. in Shanghai); wherein, the map of the virus expression vector GV492 is shown in figure 5, and the gene sequence of the c-myc ER is shown in SEQ ID NO. 1.
The method comprises the following specific steps:
1. amplification of target Gene
(1) And (3) carrying out enzyme digestion on the vector: prepare 50. mu.l of enzyme digestion system. Adding the mixed reagent, gently blowing and beating by using a pipette, mixing uniformly, centrifuging for a short time, and reacting at 37 ℃ for 3h or overnight. And (4) carrying out agarose gel electrophoresis on the vector enzyme digestion product, and recovering a target band.
(2) Obtaining a target gene fragment: artificially synthesizing a c-myc ER gene sequence, preparing a reaction system, gently blowing, uniformly mixing, centrifuging for a short time, and placing in a PCR instrument for reaction.
(3) And connecting the PCR product with a vector: preparing a reaction system, lightly blowing and uniformly mixing by using a pipette, centrifuging for a short time to avoid generating bubbles, reacting for 30min at 37 ℃, and then placing in an ice water bath for cooling for 5min and immediately converting.
(4) And (3) transformation: adding 10 μ L of the ligation reaction product into 100 μ L of competent cells, flicking the tube wall, mixing, standing on ice for 30min, thermally shocking at 42 deg.C for 90s, incubating in ice water bath for 2min, adding 500 μ L of LB medium, and shake-culturing at 37 deg.C for 1 h. Taking a proper amount of bacterial liquid, uniformly coating the bacterial liquid on a flat plate containing corresponding antibiotics, and carrying out inverted culture in a constant-temperature incubator for 12-16 h.
(5) Colony PCR identification: preparing an identification system, shaking and mixing uniformly, and centrifuging for a short time. Picking single colony in a super clean bench by using a sterile gun head to a 20 mu L identification system, blowing, uniformly mixing, and placing in a PCR instrument for reaction.
(6) Sequencing: and (3) inoculating the identified positive clone transformant into a proper amount of LB liquid culture medium containing corresponding antibiotics, culturing at 37 ℃ for 12-16h, and taking a proper amount of bacterial liquid for sequencing. And comparing and analyzing the sequencing result and the target gene sequence. The comparison result shows that: the sequencing result is completely consistent with the target sequence.
2. Plasmid extraction
Transferring the correctly sequenced bacterium liquid into 10ml LB liquid culture medium containing corresponding antibiotics, culturing overnight at 37 ℃, performing plasmid extraction by using a small-medium-amount plasmid extraction kit without endotoxin from Tiangen, and introducing the qualified plasmids into a downstream process. The detailed operation steps are as follows:
(1) collecting overnight cultured bacteria liquid in a marked 5ml centrifuge tube, centrifuging at 12000rpm for 2min, and collecting bacteria;
(2) discarding the supernatant, adding 250 μ l of cell resuspension, and fully oscillating to make the bacterial mass suspend uniformly;
(3) adding 250 μ l cell lysate, adding 10 μ l proteinase K, reversing the mixture from top to bottom for 5-6 times, and mixing gently; standing for 1-2min to make thallus cracking and clarifying;
(4) adding 350 μ l of neutralizing solution, turning upside down, mixing to completely separate out protein, and standing in ice bath for 5 min;
(5) centrifuging at 10000rpm for 10min, discarding protein, collecting supernatant in another clean sterile 1.5ml EP tube;
(6) centrifuging at 12000rpm for 5min while preparing marked recovery column, transferring the supernatant to the recovery column, centrifuging at 12000rpm for 1min, and discarding the lower layer waste liquid;
(7) adding 600 μ l of pre-prepared rinsing liquid, centrifuging at 12000rpm for 1min, discarding the lower layer waste liquid, repeating once, and allowing to idle at 12000rpm for 2min to further remove the residual rinsing liquid;
(8) transferring the recovery column to a new 1.5ml EP tube in a super clean bench, standing for 10-20min, and naturally drying;
(9) adding 95 μ l of nucleic-Free Water into the recovery column, standing for 2min, centrifuging at 12000rpm for 2min, collecting the sample, numbering, electrophoresing, measuring the concentration, and performing quality inspection.
Example 3: plasmid transfection and lentivirus harvesting
293T cells were co-transfected with plasmids. Harvesting the virus (namely unpurified cell supernatant) 48-72h after the transfection is finished, determining to obtain the high-titer lentivirus preservative solution by adopting a corresponding concentration and purification mode according to different experimental requirements, and finally determining various indexes of the lentivirus according to strict quality standards. The lentivirus particles within a certain titer range can meet most of in vivo and in vitro experimental requirements, and the process is as follows.
1. Plasmid transfection
(1) 24h before transfection, 293T cells in logarithmic growth phase were trypsinized and cell density was adjusted to about 5X10 in medium containing 10% serum6Cells/15 ml, reseeded in 10cm cell culture dishes at 37 ℃ with 5% CO2Culturing in an incubator. The cell can be used for transfection after 24 hours when the cell density reaches 70-80%;
(2) replacing the medium with a serum-free medium 2h before transfection;
(3) each prepared DNA solution (20. mu.g of GV492 vector plasmid, 15. mu.g of pHelper1.0 vector plasmid, 10. mu.g of pHelper 2.0 vector plasmid) was added to a sterilized centrifuge tube, mixed with a corresponding volume of Fugene6 transfection reagent (Boehringer Mannheim) uniformly, adjusted to a total volume of 1ml, and incubated at room temperature for 15 min;
(4) slowly dripping the mixed solutionAdding into 293T cell culture medium, uniformly mixing without blowing up cells, and mixing at 37 deg.C with 5% CO2Culturing in a cell culture box;
(5) 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;
(6) slowly adding 10% serum-containing cell culture medium 20ml, and adding 5% CO at 37 deg.C2Culturing in the incubator for 48-72 h.
2. Lentiviral concentration and purification
(1) According to the cell state, collecting the supernatant of 293T cells 48h after transfection (counted by 0 h);
(2) centrifuging at 4000g for 10min at 4 deg.C to remove cell debris;
(3) filtering the supernatant with a 0.45 μm filter in a 40ml ultracentrifuge tube;
(4) 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 ℃;
(5) after centrifugation is finished, removing supernatant, removing liquid remained on the tube wall as much as possible, adding virus preservation solution (which can be replaced by PBS or cell culture medium), and lightly and repeatedly blowing and resuspending;
(6) after full dissolution, centrifuging at high speed 10000rpm for 5min, and taking the supernatant to be packaged according to the requirement;
(7) preparing a sample to be detected.
3. Lentiviral quality detection
The main points of the quality control of lentiviruses comprise physical state detection, sterility detection and virus titer detection.
(1) Physical index detection
1) And (3) color judgment: judging by naked eyes, wherein the lentivirus preservation solution is in a pink clear liquid state;
2) and (3) viscosity judgment: slowly sucking 50 mul of lentivirus preservation liquid by using a 20-200 mul specification pipettor, and having no obvious viscous feeling or liquid absorption hysteresis phenomenon;
(2) and (3) sterility detection: the virus is added into 293T cells for verification, microscopic examination is carried out after normal culture is carried out for 24h, the condition of any bacteria and fungus pollution is avoided, meanwhile, no obvious particles exist in cell gaps according to an empty cell group, and a culture medium is clear and transparent.
(3) Titer detection and analysis: absolute quantitative qPCR method
The lentivirus can integrate the 5 'LTR-3' LTR region of the virus into the host genome for stable expression, and the virus infects the tool cell 293T, so that the virus characteristic single copy gene A and the host characteristic single copy gene B in the tool cell 293T genome are detected by an absolute quantitative method. And (3) calculating the average number of infected virus particles in each cell, multiplying the average number by the number of cells in each hole, and dividing the number by the infection amount to obtain the titer of the virus sample.
Calculating the formula:
qPCR titer (TU/ml) ═ N × C/V
N is the number of cells in the corresponding well in the 24-well plate at the time of infection;
c (the number of lentiviruses contained in each cell) ═ a copy number/B copy number) × 2;
v-the volume of lentivirus (ml) infected in the corresponding well.
The experimental steps are as follows:
1) preparing a standard substance: constructing a plasmid standard substance A containing a lentivirus genome conserved sequence a and a plasmid standard substance B containing a single copy gene B in a 293T genome of a tool cell; concentration of 1X1010copy/μ l, subpackaged and stored at-80 deg.C for a long time.
2) Designing and preparing a primer: qPCR primers are designed aiming at the plasmid standard products A and B respectively, and 10 mu M of primer working solution is prepared.
3) Sample preparation:
a) 24h before infection, 293T cells were cultured in 24-well plates at a density of 5X104cell/well.
b) During infection, blank control cells in 2-3 wells are collected, the total number of the cells N infected in each well is counted, the volume of virus infected in each well is V ml, and each virus infects 3 multiple wells.
c) 24h post infection, 1000. mu.l of complete medium was added to each well and handled carefully without blowing up the cells.
d) After 72h of infection, the supernatant was aspirated and photographed by fluorescence; simultaneously, the cells in the wells are collected separately.
e) The genome of the cells is extracted and collected by a Tiangen cell and blood genome extraction kit.
4) Diluting a standard product:
10 fold gradient dilution method at 109、108、107、106、105、104、103、102、101And (3) diluting the quality standard substances A and B and the sample to be detected in a gradient manner.
5) And (3) preparing a PCR reaction system.
6) And (3) PCR reaction:
the procedure was set to two step Real-Time quantitation. Pre-denaturation 95 ℃ for 15s, followed by denaturation 95 ℃ for 5s and annealing extension 60 ℃ for 30s for 40 cycles in each step. Each time reading the absorbance value during the extension phase. After the PCR is finished, a dissolution curve is made, denaturation is carried out for 1min at 95 ℃, and then cooling is carried out to 60 ℃ for 1min, so that the DNA double strands are fully combined. Starting at 60 ℃, each step was increased by 0.5 ℃ and held for 30s while absorbance was read.
7) Titre results
According to the calculation formula: qPCR titer (TU/ml) ═ N × C/V, the average titer of the samples was calculated.
Example 4: construction of conditionally-immortalized human neural stem cells
Culturing human neural stem cells by using a neural stem cell complete medium, and then adding the lentivirus prepared by the method into the human neural stem cells at a virus titer with the multiplicity of infection (MOI) of 5 so as to infect the human neural stem cells, thereby constructing recombinant human neural stem cells over-expressing c-myc ER. As the virus vector has GFP labels and resistance, the recombinant human neural stem cells with high expression are screened by a resistance culture medium containing puromycin. Then the infection efficiency is judged by comparing the cell condition under a fluorescence microscope with the cell condition under a common light mirror.
The method comprises the following specific steps:
(1) preparation of a Density of 1X10 Using complete Medium5Individual/ml human neural stem cell suspension at 4X 104One/ml was inoculated into each well of a six-well plate.
(2) And after the cells adhere to the wall, changing the liquid of the cells, adding a corresponding amount of c-myc ER lentivirus according to the MOI (average molar equivalent) of the cells to 5, culturing at 37 ℃ for 12-16h, and replacing a complete culture medium for continuous culture.
(3) About 72 hours after infection, the infection efficiency was observed. The infection efficiency of 80% is the optimal infection efficiency.
The infection condition of the recombinant human neural stem cells is shown in figure 6, wherein A: a picture of the recombinant human neural stem cells under a common optical microscope; b: and (3) pictures of the recombinant human neural stem cells under a fluorescence microscope. Therefore, the c-myc ER gene is successfully expressed in the recombinant human neural stem cell.
After the condition immortalization human neural stem cells are established, the karyotype of the human neural stem cells is analyzed. Karyotype refers to the phenotype of a genome in metaphase of mitosis, including chromosome number, size, morphological features, and the like. And performing measurement calculation on the constructed conditionally-immortalized human neural stem cell chromosomes, and then performing grouping, queuing, pairing and morphological analysis.
The results are shown in FIG. 7, and the constructed conditionally-immortalized human neural stem cell has normal karyotype.
Example 5: method for accelerating immortalization human neural stem cell proliferation rate through drug-induced condition
1. Verification of drug-inducible c-myc protein activation by downstream gene expression
Activating the activity of the c-myc protein in the cells by adding 4-hydroxy tamoxifen into the recombinant neural stem cells, and detecting the transcription condition of downstream genes by a PCR method, wherein the downstream genes are selected from cad, mrdb, ord, rccl and rcl.
The method comprises the following specific steps:
(1) preparing a neural stem cell complete culture medium containing 100nM 4-hydroxyttamoxifen;
(2) the recombinant human neural stem cells are divided into two groups, the neural stem cell complete culture medium is added into a control group, and the neural stem cell complete culture medium containing 100nM 4-hydroxy tamoxifen is added into an experimental group. After culturing for 4 days, PCR is carried out to detect the transcription condition of the downstream gene of the c-myc.
(3) Respectively taking the neural stem cells to extract mRNA, carrying out reverse transcription to obtain cDNA, and carrying out PCR detection. The detection index is c-myc downstream activating genes, including cad, mrdb, ord, rccl, rcl and other sequences.
The expression levels of cad, mrdb, ord, rccl and rcl of the 4-hydroxytamoxifen-added recombinant neural stem cells (left side) were all significantly higher than those of the 4-hydroxytamoxifen-added recombinant neural stem cell experimental group (right side), as shown in fig. 8.
2. Evaluation by fusion area of cells
After the conditionally immortalized human neural stem cells are cultured in an adherent manner, the cells are placed in a long-term monitoring device to monitor the dynamic process of cell proliferation, the growth trend of the cells is detected according to the adherent fusion area of the recombinant neural stem cells, and the difference of the cell proliferation rate of the experimental group added with 4-hydroxy tamoxifen and the experimental group not added with 4-hydroxy tamoxifen is compared.
The method comprises the following specific steps:
dividing human neural stem cells to be detected into two groups, namely (A) recombinant neural stem cells and adding a neural stem cell complete culture medium; (B) recombinant neural stem cells were added to neural stem completion medium containing 100nM 4-hydroxy tamoxifen. Two groups of neural stem cells are respectively inoculated in a six-hole plate, each group of cells is inoculated in three holes, and the inoculation density is 5 multiplied by 105A hole. After inoculation, the cells were monitored in a long-term monitoring apparatus for 4.5 days. And comparing the proliferation rates of the two groups of neural stem cells according to the fusion area rate of the cells.
The proliferation efficiency ratio of the recombinant neural stem cells added with 4-hydroxy tamoxifen and the conditionally-immortalized human neural stem cells without 4-hydroxy tamoxifen is shown in fig. 9, and further proves that the proliferation efficiency of the conditionally-immortalized human neural stem cells added with 4-hydroxy tamoxifen is obviously different from that of the conditionally-immortalized human neural stem cells without 4-hydroxy tamoxifen, and the conditionally-immortalized human neural stem cells added with 4-hydroxy tamoxifen have a faster proliferation rate.
3. Detection of neural stem cell proliferation by EdU labeling
EdU is a thymidine analog, which can penetrate into replicating DNA molecules instead of thymidine (T) during cell proliferation, and detects DNA replication activity by specific reaction based on EdU with fluorescent dye, and can accurately reflect the proliferation of neural stem cells by detecting EdU label.
The method comprises the following specific steps:
dividing human neural stem cells to be detected into four groups, namely (A) adding a neural stem cell complete culture medium into the neural stem cells; (B) adding neural stem cells to neural stem cell-complete medium containing 100nM 4-hydroxy tamoxifen; (C) adding the conditioned immortalized human neural stem cells into a neural stem cell complete culture medium; (D) conditionally-immortalized human neural stem cells were added to neural stem-complete medium containing 100nM 4-hydroxyttamoxifen. Four groups of neural stem cells are inoculated in a confocal small dish, and the inoculation density is 1x105Perwell, 24h later EdU dye (purchased from Millipore, #17-10525) was added and fluorescent staining was performed after 48h of incubation.
Fixing the neural stem cells with 4% paraformaldehyde for 15min before staining, washing with PBS, and incubating the cells with 0.05% triton X-100 for 20min to make the cell membrane permeable. And finally, dyeing by using an EdU dye corresponding to a fluorescent reagent, and observing under a fluorescent microscope after dyeing is finished. According to the condition that the EdU dye marks the neural stem cells, the proliferation rate difference of the four groups of neural stem cells is compared.
The proliferation rate pairs of four groups of neural stem cells are shown in fig. 10, so that the proliferation efficiency of the conditionally-immortalized human neural stem cells added with 4-hydroxy tamoxifen is remarkably different from that of the other three groups of neural stem cells, the proliferation efficiency is fastest, and the proliferation rate of the other three groups of neural stem cells is not obviously different.
Example 6 preparation of cell Membrane nanovesicles derived from conditionally-immortalized human neural Stem cells
1. Preparation of conditionally-immortalized neural stem cell-derived cell membrane nano-vesicles
Subjecting conditionally-immortalized human neural stem cells to expanded culture, digesting the cells with accutase, centrifuging and collecting 1x107The cells were resuspended in 5mL of PBS, and the cell suspension was passed through a 10-. mu.m, 5-. mu.m, 1-. mu.m pore size acetic acid membrane (Whatman, 110615, 110613, 800319) in sequence using an extruder (Sigma, 610000-1EA), and repeatedly extruded 8 to 12 times. Collecting the suspension after membrane extrusion, centrifuging at 4 ℃ for 15min at 3000g to remove cell fragments, taking the supernatant, centrifuging at 4 ℃ for 15min at 20000g, discarding the supernatant, and resuspending the precipitate with 200-fold 500 mu L PBS to obtain the conditionally-immortalized human neural stem cell membrane-derived cell membrane nano vesicles; the whole process is placed on ice or in a low-temperature environment to prevent protein denaturation.
2. Surface marker characterization of neural stem cell nanovesicles
WB is used for detecting the human nerve stem cells and the nano vesicle surface markers from the conditionally-immortalized human nerve stem cells, 30 mu g of cell membrane nano vesicle suspension is taken and added into 5 XLoadding Buffer boiling water for treatment for 5min, and 10% SDS-PAGE gel electrophoresis is used for separation. Transferring to PVDF membrane, sealing with 5% skimmed milk for 1h, adding exosome marker molecule antibody anti-CD9 (diluted 1: 1000) and anti-TSG101 antibody (diluted 1: 1000), and incubating at 4 deg.C overnight; the secondary antibody is incubated for 60min at room temperature in the dark, and is subjected to ECL development photographing after 1 XPBST rinsing.
The results are shown in fig. 11, and it can be seen that the human neural stem cells and the conditionally-immortalized human neural stem cell membrane nanovesicle surface markers CD9 and TSG101 are both positive.
3. Characterization of particle size and quantity of neural stem cell nano-vesicles
And (2) detecting the diameter distribution and the number of the prepared cell membrane nano vesicles derived from the conditionally immortalized human neural stem cells by utilizing Nanoparticle Tracking Analysis (NTA), taking 1ml of cell membrane nano vesicles, pushing the cell membrane nano vesicles into the NTA by using a syringe injector, and detecting the particle diameter and the number of the cell membrane nano vesicles.
As a result, as shown in FIG. 12, it can be seen that the average particle size of the conditionally-immortalized human neural stem cell-derived cell membrane nanovesicles was 182.4 nm. Calculated, 1.2 x107Preparation of 2.61X 10 cells11The single cell can prepare 21750 cell membrane nano vesicles, and the method for preparing the cell membrane nano vesicles is high in yield and suitable for industrial production.
4. Morphological characterization of conditionally-immortalized human neural stem cell membrane nanovesicles
Performing morphological detection on the prepared cell membrane nanovesicle by using a transmission electron microscope, performing ultrasonic treatment on the cell membrane nanovesicle suspension for 20min by 10 mu L, fixing the cell membrane nanovesicle suspension for 30min at room temperature by using isovolumetric 4% paraformaldehyde, settling the cell membrane nanovesicle suspension on a copper net, dyeing the cell membrane nanovesicle suspension for 5min by using phosphotungstic acid, removing redundant liquid, drying the cell membrane nanovesicle suspension, and collecting and observing the morphology of the cell membrane nanovesicle by using a TEM.
As a result, as shown in fig. 13, it was observed that the cell membrane nanovesicles are all lipid bilayer structures, and are circular or cup-shaped.
Example 7 conditionally-immortalized human neural Stem cell-derived Membrane nanovesicles repair neuronal damage
1. Construction of model of neuron hypoxia injury
Selecting well-grown mouse neuron cells (HT22), dividing into control group and anoxic group, after passage adherence, respectively using DMEM complete culture medium (BI,06-1055-57-1ACS) and HBSS (GIBCO, 24010043) to exchange liquid, placing in 37 deg.C cell culture box (5% CO)2And 95% N2) The culture was carried out for 10 hours.
2. Detection of model of neuronal hypoxic injury
Using Image-iTTMGreen hypoxia monitoring reagent (Thermo, I14833) was used for the detection of neuronal hypoxia model. The reagent is a novel, fixable, fluorescent compound used to determine hypoxia in living cells. Living cells do not fluoresce in an environment with normal oxygen concentrations, but fluoresce when oxygen levels are reduced. Image-iT Green hypoxia monitoring agents can maintain their fluorescence when cells/tissues return to normal oxygen levels.
The method comprises the following specific steps:
(1) 2x10 to5Individual HT22 cells were plated overnight in 22mm confocal dishes.
(2) The hypoxia monitoring reagent stock solution was diluted in the medium at a final concentration of 1 μm, and the hypoxic group and the control group were incubated together at 37 ℃ for 30 min.
(3) Control cells were placed in normal culture conditions (5% CO)2) Culturing at 37 deg.C for 10 hr, and placing the anoxic group in anoxic equipment (5% CO)2And 95% N2) Culturing at 37 deg.C for 10 h.
(4) Imaging was performed after 10 hours using a fluorescence microscope.
As a result, as shown in fig. 14, the success of the hypoxia model was found from the Control (Control) and hypoxia (OGD) imaging effect graphs.
3. Condition immortalized human neural stem cell-derived cell membrane nano-vesicle for treating hypoxia injury neurons
Using eBioscienceTMAnnexin V-FITC Apoptosis Detection Kit (Thermo, BMS500FI-100) tests the effect of vesicles on treating hypoxia-damaged neurons. Annexin v (annexin v) is a series of calcium-dependent phospholipid binding proteins that bind to Phosphatidylserine (PS) to identify apoptotic cells. In healthy cells, PS is predominantly located on the cytosolic side of the plasma membrane. Following the onset of apoptosis, the asymmetric distribution of PS in the phospholipid bilayer gradually disappeared and transferred to the outer cell membrane, which was detected by fluorescently labeled Annexin V. In the early stage of apoptosis, the plasma membrane can prevent active dyes such as Propidium Iodide (PI) from entering cells, so that only cells showing Annexin V staining positive (PI negative) are in the early stage of apoptosis. In the late stage of apoptosis, Annexin V binds to cytosolic PS due to loss of integrity of the cell membrane, and the cell begins to take up PI. Annexin V staining is matched with PI for use, and can be widely applied to analysis and identification of the apoptosis stage by a flow cytometer.
(1) Neurons were divided into three groups: control group (Control), anoxic group (OGD) and post-anoxic condition immortalized human neural stem cell derived cell membrane nanovesicle treatment group (NVs). The control group was normal cultured neurons and the hypoxic group was in hypoxic equipment (5% CO)2And 95% N2) Culturing at 37 deg.C for 10 hr, and treating with vesicle after anoxiaAfter hypoxia, the neurons were incubated with vesicles (final vesicle concentration of 20. mu.g/ml) for 24 h.
(2) Neurons from the control, hypoxic and post-hypoxic vesicle-treated groups were washed once with PBS before testing.
(3) The blank cells were resuspended at a cell density of 2-5X 10 using 200. mu.l Binding Buffer (1X)5/mL。
(4) The stained cells were resuspended using 5. mu.L Annexin V-FITC and 195. mu.L Binding Buffer (1X) at a cell density of 2-5X 105mL, incubated at room temperature for 10min in the dark.
(5) After incubation, 10. mu.l of PI (20. mu.g/ml) was added to the cell suspension.
(6) Apoptosis analysis was performed by flow cytometry.
The flow-type apoptosis analysis results of the three groups of neurons are shown in fig. 15, and it can be seen that the early apoptosis of the cell membrane nano vesicle treatment group neurons is obviously reduced (Annexin-V positive and PI negative) compared with the hypoxia group.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
SEQUENCE LISTING
<110> Dalian Stem cell and precision medical Innovation research institute
<120> conditionally-immortalized human neural stem cell-derived cell membrane nano vesicle preparation as well as preparation method and application thereof
<130> 20210805
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 2428
<212> DNA
<213> Artificial sequence
<400> 1
gtttttggct tttttgttag acgaagcttg ggctgcaggt cgactctaga ggatcccgcc 60
accatgcccc tcaacgttag cttcaccaac aggaactatg acctcgacta cgactcggtg 120
cagccgtatt tctactgcga cgaggaggag aacttctacc agcagcagca gcagagcgag 180
ctgcagcccc cggcgcccag cgaggatatc tggaagaaat tcgagctgct gcccaccccg 240
cccctgtccc ctagccgccg ctccgggctc tgctcgccct cctacgttgc ggtcacaccc 300
ttctcccttc ggggagacaa cgacggcggt ggcgggagct tctccacggc cgaccagctg 360
gagatggtga ccgagctgct gggaggagac atggtgaacc agagtttcat ctgcgacccg 420
gacgacgaga ccttcatcaa aaacatcatc atccaggact gtatgtggag cggcttctcg 480
gccgccgcca agctcgtctc agagaagctg gcctcctacc aggctgcgcg caaagacagc 540
ggcagcccga accccgcccg cggccacagc gtctgctcca cctccagctt gtacctgcag 600
gatctgagcg ccgccgcctc agagtgcatc gacccctcgg tggtcttccc ctaccctctc 660
aacgacagca gctcgcccaa gtcctgcgcc tcgcaagact ccagcgcctt ctctccgtcc 720
tcggattctc tgctctcctc gacggagtcc tccccgcagg gcagccccga gcccctggtg 780
ctccatgagg agacaccgcc caccaccagc agcgactctg aggaggaaca agaagatgag 840
gaagaaatcg atgttgtttc tgtggaaaag aggcaggctc ctggcaaaag gtcagagtct 900
ggatcacctt ctgctggagg ccacagcaaa cctcctcaca gcccactggt cctcaagagg 960
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cgaaaatgca ccagccccag gtcctcggac accgaggaga atgtcaagag gcgaacacac 1140
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ttgttggatg ctgaaccgcc catgatctat tctgaatatg atccttctag acccttcagt 1560
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ctcgagtgtg cctggctgga gattctgatg attggtctcg tctggcgctc catggaacac 1740
ccggggaagc tcctgtttgc tcctaacttg ctcctggaca ggaatcaagg taaatgtgtg 1800
gaaggcatgg tggagatctt tgacatgttg cttgctacgt caagtcggtt ccgcatgatg 1860
aacctgcagg gtgaagagtt tgtgtgcctc aaatccatca ttttgcttaa ttccggagtg 1920
tacacgtttc tgtccagcac cttgaagtct ctggaagaga aggaccacat ccaccgtgtc 1980
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cagcagcatc gccgcctagc tcagctcctt ctcattcttt cccatatccg gcatatgagt 2100
aacaaacgca tggagcatct ctacaacatg aaatgcaaga acgtggtacc cctctatgac 2160
ctgctcctgg agatgttgga tgcccaccgc cttcatgccc cagccagtcg catgggagtg 2220
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Claims (10)
1. A preparation method of conditionally-immortalized human neural stem cell-derived cell membrane nanovesicles is characterized by mainly comprising the following steps:
(1) constructing conditionally-immortalized human neural stem cells by a genetic engineering means, wherein the proliferation speed of the conditionally-immortalized human neural stem cells is accelerated under the action of a specific medicament;
(2) adding the specific medicine into the culture system of the conditionally-immortalized human neural stem cells obtained in the step (1), improving the proliferation speed of the conditionally-immortalized human neural stem cells, and culturing to obtain a large batch of conditionally-immortalized human neural stem cells;
(3) promoting the conditionally-immortalized human neural stem cell membranes obtained in the step (2) to be fused again by a physical extrusion method, and obtaining the cell membrane nano vesicles with maternal cell characteristics in a large scale.
2. The method for producing according to claim 1, wherein the conditionally-immortalized human neural stem cell in step (1) expresses an estrogen-inducible c-myc system.
3. The method of claim 2, wherein the estrogen-inducible c-myc system is formed by fusing c-myc to the estrogen receptor ER.
4. The method according to claim 2, wherein the estrogen-inducible c-myc system has the nucleotide sequence shown in SEQ ID NO 1.
5. The method for preparing a conditionally-immortalized human neural stem cell according to any one of claims 2 to 4, wherein the step (1) comprises introducing a gene encoding an estrogen-inducible c-myc system directly into the host cell or introducing a recombinant virus carrying a gene encoding an estrogen-inducible c-myc system into the host cell by viral transfection.
6. The method of claim 1, wherein the specific drug comprises tamoxifen, 4-hydroxytamoxifen; the specific drug has reversibility to the regulation of the proliferation rate of the immortalized human neural stem cells.
7. The method according to claim 6, wherein the final concentration of the specific drug in the culture system is 10 to 1000 nM.
8. The preparation method according to claim 1, wherein the specific operation of the physical extrusion method in step (3) comprises passing the cell suspension of conditionally-immortalized human neural stem cells through a filter membrane with a pore size of 10 μm, 5 μm, and 1 μm in sequence by an extruder, repeatedly extruding for 5-20 times, collecting the obtained suspension, centrifuging at 0-4 ℃ for 5-30 min at 1000-5000 g, collecting the supernatant, centrifuging at 0-4 ℃ for 5-30 min at 10000-30000 g, discarding the supernatant, and resuspending the obtained precipitate with a buffer solution to obtain the cell membrane nanovesicles.
9. A cell membrane nanovesicle produced by the production method according to any one of claims 1 to 8.
10. Use of the cell membrane nanovesicles of claim 9 in the preparation of a medicament for the treatment of a central nervous system disorder.
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