WO2023010637A1 - 一种条件永生化人神经干细胞来源细胞膜纳米囊泡制剂及其制备方法和应用 - Google Patents

一种条件永生化人神经干细胞来源细胞膜纳米囊泡制剂及其制备方法和应用 Download PDF

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WO2023010637A1
WO2023010637A1 PCT/CN2021/116164 CN2021116164W WO2023010637A1 WO 2023010637 A1 WO2023010637 A1 WO 2023010637A1 CN 2021116164 W CN2021116164 W CN 2021116164W WO 2023010637 A1 WO2023010637 A1 WO 2023010637A1
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neural stem
human neural
cell
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stem cells
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刘晶
王亮
王佳一
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大连干细胞与精准医学创新研究院
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Definitions

  • the invention belongs to the technical field of cell engineering and genetic engineering, and in particular relates to a conditionally immortalized human neural stem cell-derived cell membrane nanovesicle preparation and its preparation method and application.
  • neural stem cells as seed cells derived from the central nervous system, have a more effective role in the treatment of nervous system diseases.
  • a large number of preclinical studies have shown that neural stem cell transplantation, as a potential treatment, plays an important role in central nervous system diseases through various mechanisms such as producing neurotrophic factors, reducing neuroinflammation, enhancing neuroplasticity and cell replacement.
  • neural stem cell transplantation plays an important role in central nervous system diseases through various mechanisms such as producing neurotrophic factors, reducing neuroinflammation, enhancing neuroplasticity and cell replacement.
  • only a few clinical research results have been published so far, and the clinical application of neural stem cells is facing unprecedented challenges.
  • Neural stem cell-derived extracellular vesicles provide a particularly attractive alternative to cell transplantation for basic therapy, and unlike cell therapy, the probability of safety issues after administration of EVs is almost zero, because they Nonnucleated, unable to replicate, degrades quickly after entering the cell.
  • NSC-mediated functional recovery is mainly attributed to the trophic support provided by NSC 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 treatment. Therefore, cell-free therapy has attracted great interest.
  • the purpose of the present invention is to provide a cell membrane nanovesicle preparation derived from conditionally immortalized human neural stem cells and its preparation method, and to construct a complete system from cells to subsequent preparations of cell derivatives to solve the problem of human Neural stem cell derivatives are facing the problem of difficult large-scale production encountered in clinical translation.
  • the object of the invention is to realize in the following way:
  • the present invention first transforms human neural stem cells through genetic engineering technology, constructs conditionally immortalized human neural stem cells, accelerates its proliferation rate, enables large-scale cultivation, and ensures the stability of the preparation source of cell membrane nanovesicle preparations.
  • a method for preparing a conditionally immortalized human neural stem cell-derived cell membrane nanovesicle preparation mainly comprising the following steps:
  • conditional immortalized human neural stem cells by means of genetic engineering, the proliferation speed of the conditionally immortalized human neural stem cells is accelerated under the action of specific drugs, and they can be expanded and cultured in large quantities;
  • step (2) Add the specific drug to the conditional immortalized human neural stem cell culture system obtained in step (1) to increase the proliferation rate of the conditional immortalized human neural stem cell, and obtain a large amount of conditional immortalized human neural stem cells after culturing neural stem cells;
  • step (3) Promoting the membrane refusion of the conditionally immortalized human neural stem cells obtained in step (2) by a physical extrusion method to obtain cell membrane nanovesicles with characteristics of parent cells.
  • the immortalized human neural stem cells described in step (1) express the c-myc ER system (estrogen-inducible c-myc system).
  • the c-myc ER system is formed by fusing c-myc and estrogen receptor ER.
  • nucleotide sequence of the c-myc ER system is shown in SEQ ID NO:1.
  • the specific process of constructing conditional immortalized human neural stem cells described in step (1) is to directly introduce the gene encoding the c-myc ER system into the host cell or to introduce the gene encoding the c-myc ER system into the host cell through viral transfection.
  • the recombinant virus of the gene of -myc ER system is introduced into the host cell.
  • the ligand binding region of the estrogen receptor of the c-myc ER system expressed by the conditionally immortalized human neural stem cells contains a point mutation (G521R).
  • the specific drug includes tamoxifen and 4-hydroxytamoxifen.
  • the final concentration of the specific drug in the culture system is 10-1000 nM.
  • the regulation of the proliferation rate of the immortalized cells by the specific drug is reversible.
  • the drug is added to the immortalized cells, the reactive proliferation rate of the immortalized cells is accelerated, and when the addition of the drug is stopped, the immortalized cells will be immortalized.
  • the proliferation rate of the cultured cells will gradually return to the original level.
  • the specific operation steps of the physical extrusion method in step (3) include passing the cell suspension of the conditionally immortalized human neural stem cells through an extruder with an aperture size of 10 ⁇ m, 5 ⁇ m, and 1 ⁇ m in sequence.
  • the filter membrane was repeatedly extruded 5-20 times, and the resulting suspension was collected, centrifuged at 0-4°C, 1000-5000g for 5-30min, and the supernatant was taken, and centrifuged at 0-4°C, 10000-30000g for 5-30min. The supernatant was discarded, and the resulting pellet was resuspended with buffer to obtain cell membrane nanovesicles.
  • Another aspect of the present invention provides a cell membrane nanovesicle prepared by the above preparation method.
  • Another aspect of the present invention provides the application of the above-mentioned cell membrane nanovesicles in the preparation of drugs for treating diseases of the central nervous system.
  • conditional immortalized human neural stem cell-derived cell membrane nanovesicle preparation and its preparation method that can be prepared on a large scale in the present invention were first developed and proposed by the inventors.
  • Human neural stem cells are conditionally immortalized through genetic engineering technology, It can be expanded and cultured in large quantities in vitro, thereby providing a stable source of preparation for human neural stem cell derivative preparations.
  • cell membrane nanovesicles derived from conditionally immortalized human neural stem cells with characteristics of maternal cells were prepared on a large scale by physical extrusion to promote cell membrane refusion.
  • a complete system from cells to subsequent preparations of cell derivatives has been constructed, which can solve the problem of large-scale production of preparations encountered in the clinical transformation of human neural stem cell derivatives.
  • the preparation method of the conditionally immortalized human neural stem cell-derived cell membrane nanovesicle preparation that can be prepared on a large scale in the present invention has the characteristics of high yield, simple operation, and strong repeatability.
  • the conditional immortalization construction scheme selected in the genetic engineering modification of human neural stem cells can regulate its proliferation rate through the addition of exogenous drugs, ensuring the safety of the parental cells from which the preparation is derived.
  • the present invention provides a complete set of preparation solutions from cells to cell derivative preparations, which will be clinically oriented to subsequent preparations of cell derivatives derived from other cells. Translational research provides references.
  • 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 the optical microscope picture of the human neural stem cells of different generations (the 2nd generation, 4th generation and 8th generation) in the embodiment of the present invention 1;
  • Fig. 3 is a picture of specific marker identification of human neural stem cells in Example 1 of the present invention.
  • Fig. 4 is the detection picture of human neural stem cell inducing differentiation ability in embodiment 1 of the present invention.
  • Figure 5 is a map of the lentiviral expression vector encoding the estrogen-inducible c-myc system in Example 1 of the present invention
  • Example 6 is a picture of human neural stem cells under a fluorescence microscope after lentivirus infection in Example 1 of the present invention
  • Figure 7 is a picture of the karyotype analysis of conditionally immortalized human neural stem cells of the present invention.
  • Fig. 8 is a picture of downstream gene transcription of recombinant human neural stem cells in Example 1 of the present invention after adding 4-hydroxytamoxifen, wherein 4-OHT is the drug-dosed group, and un-4-OHT is the complete medium experimental group;
  • Figure 9 is a picture of the change in proliferation rate of recombinant human neural stem cells in Example 1 of the present invention without or with 4-hydroxytamoxifen, wherein 4-OHT is the drug-dosed group, and un-4-OHT is the complete medium test group;
  • Fig. 10 is the human neural stem cell of the present invention and recombinant human neural stem cell EdU labeling detection proliferation experiment;
  • Figure 11 is a Western Blot diagram of conditionally immortalized human neural stem cell-derived cell membrane nanovesicles in Example 6 of the present invention.
  • Figure 12 is a transmission electron micrograph of the conditionally immortalized human neural stem cell-derived cell membrane nanovesicles in Example 6 of the present invention.
  • Figure 13 is a particle size distribution diagram of conditionally immortalized human neural stem cell-derived cell membrane nanovesicles in Example 6 of the present invention.
  • Figure 14 is the construction of the neuron hypoxic injury model in Example 7 of the present invention.
  • Fig. 15 shows the apoptosis after treatment with the immortalized human neural stem cell-derived cell membrane nanovesicles in Example 7 of the present invention.
  • the experimental methods used are conventional methods, and the materials and reagents used can be purchased from biological or chemical reagent companies.
  • the invention improves the proliferative ability of human neural stem cells (NSCs) by transforming them. First, culture and identify human neural stem cells.
  • the culture method of human neural stem cells is suspension culture in the complete medium of neural stem cells (purchased from Stem cell technology, catalog#05751).
  • the specific operation method is:
  • the primary extraction and preparation of neural stem cells is carried out in a biological safety cabinet, and the operation process requires strict aseptic operation. Take a sterile petri dish with a diameter of 100 mm, pour 10 mL of sterile normal saline, and place the intact aborted fetal tissue (in line with the "Guiding Principles of Human Embryonic Stem Cell Research Ethics") in normal saline solution for repeated washing until the normal saline is clear and transparent .
  • Use sterilized surgical scissors and tweezers to peel off the skin of the head of the aborted fetus, cut open the skull to fully expose the brain tissue, use surgical tweezers to gently tear off the pia mater around the brain tissue, and peel off the blood vessels as much as possible.
  • the fetal brain tissue was completely taken out, put into a new Petri dish filled with sterile saline, and the cerebral cortex was carefully separated. Prepare a 35mm sterile Petri dish, put it into the neural stem complete medium, put the isolated cortical tissue into the medium and divide it into 1mm tissue pieces. Transfer the complete medium containing the tissue pieces to a 50mL centrifuge tube with a pipette, pipette gently 30 times, put it in a 37°C incubator, let it stand for 10 minutes, take the supernatant into a new 50mL centrifuge tube, and centrifuge at 300g for 5 minutes, discard the supernatant.
  • the cells After inoculation, inspect and observe under an ordinary optical microscope to confirm that the cells are roughly evenly distributed in each culture container. If the cells are not evenly distributed, shake the container again. Record cell status and abnormalities.
  • the cells after microscopic examination were placed in a carbon dioxide incubator at a culture temperature of 37°C and a CO 2 concentration of 5%. 2-3 days after cell inoculation, observe under the microscope, record the growth state of the cells, and carry out rehydration, the rehydration volume is 2-3mL. After that, the cells were replaced every 3 days, and the primary cell suspension culture was carried out after extraction. When the neurospheres grew to 100-150 ⁇ m, the neural stem cells were passaged.
  • the specific markers of human neural stem cells of the present invention are identified by flow cytometry.
  • the specific operation steps are: digest the cells with Accutase enzyme, add neural stem cell complete medium to blow the neural stem cells
  • the obtained neural stem cells were fixed with 4% paraformaldehyde for 15 minutes, centrifuged at 300g for 5min, discarded the supernatant, then added 2mL 1 ⁇ PBS, mixed well, centrifuged again at 300g for 5min, discarded the supernatant, Add 1 ⁇ PBS to resuspend to prepare single cell suspension (cell density is 1 ⁇ 10 6 /mL).
  • the results of the identification of human neural stem cell-specific markers are shown in Figure 3.
  • the results show that the human neural stem cells cultured in the present invention highly express Nestin and Sox2 without differentiation, and the human neural stem cells before line establishment are normal neural stem cells.
  • the human neural stem cells of the present invention are tested for their differentiation ability.
  • the specific operation steps are as follows: culture neural stem cells in a matrigel-coated six-well plate at 4 ⁇ 10 5 cells/well, and replace neural stem cell proliferation medium with neural stem cell differentiation medium (purchased from Stem cell technology, # 05752), add 2ml to each well. Change the differentiation medium every 2 to 3 days, and after 10 days of differentiation induction culture, perform immunofluorescence staining identification on the CQ1 laser confocal high-content cell sorting system (YOKOGAWA, CQ1) to verify that the extracted human neural stem cells have neurotropism. The ability of the three major nerve cells in the system to differentiate.
  • FIG. 4 The test results of human neural stem cell induction and differentiation ability are shown in Figure 4.
  • A Cell morphology observed under ordinary light microscope on the 2nd and 4th day of neural stem cell induction and differentiation;
  • B Immunofluorescent staining of neurons: mouse monoclonal antibody beta III Tubulin (Tuj1) and DAPI staining;
  • C oligodendrocyte immunofluorescence staining: rabbit polyclonal antibody Oligodendrocyte Specific Protein and DAPI staining;
  • D astrocyte immunofluorescence staining: rabbit polyclonal antibody GFAP and DAPI staining ; It further proves that the human neural stem cells extracted by the present invention have the ability to differentiate into the three major nerve cells of the nervous system.
  • Embodiment 2 Construction of expression vector
  • cells are transformed by infecting host cells with lentivirus. Therefore, an expression vector comprising the estrogen-inducible c-myc system was first constructed, and the c-myc ER gene was constructed in a lentiviral vector, and the lentiviral expression vector used was GV492 (purchased from Shanghai Jikai Gene Chemical Technology Co., Ltd.) Wherein, the viral expression vector GV492 map is shown in Figure 5, and the c-myc ER gene sequence is shown in SEQ ID NO:1.
  • Carrier enzyme digestion Prepare 50 ⁇ l enzyme digestion system. Add the mixed reagents, gently blow and mix with a pipette, centrifuge briefly, and place at 37°C for 3 hours or overnight. Perform agarose gel electrophoresis on the digested product of the vector to recover the target band.
  • Colony PCR identification Prepare an identification system, shake and mix, and briefly centrifuge. In the ultra-clean workbench, use a sterile tip to pick a single colony into a 20 ⁇ L identification system, mix it by pipetting, and place it in a PCR machine for reaction.
  • Example 3 Plasmid transfection and lentivirus harvesting
  • 293T cells were co-transfected with plasmids.
  • the quality standard measures various indicators of lentivirus. Lentiviral particles within a certain titer range can meet the needs of most in vivo and in vitro experiments, and the process is as follows.
  • the quality control points of lentivirus include physical state detection, sterility detection and virus titer detection.
  • the lentivirus can integrate the 5'LTR-3'LTR region of the virus into the host genome for stable expression.
  • the virus characteristic single copy gene A and host characteristics in the tool cell 293T genome are detected by absolute quantitative method Single copy gene B. Calculate the average number of infected virus particles per cell, multiply by the number of cells per well, and divide by the amount of infection to obtain the titer of the virus sample.
  • N the number of cells in the corresponding wells of the 24-well plate at the time of infection
  • V lentivirus volume (ml) infected in the corresponding well.
  • Standard product preparation construct plasmid standard A containing the conserved sequence a of the lentiviral genome and plasmid standard B containing a single copy gene b in the tool cell 293T genome; °C long-term preservation.
  • MOI multiplicity of infection
  • FIG. 6 The infection situation of the recombinant human neural stem cells of the present invention is shown in Figure 6, A: the picture of the recombinant human neural stem cells under an ordinary optical microscope; B: the picture of the recombinant human neural stem cells under a fluorescent microscope.
  • A the picture of the recombinant human neural stem cells under an ordinary optical microscope
  • B the picture of the recombinant human neural stem cells under a fluorescent microscope.
  • conditionally immortalized human neural stem cells After the conditionally immortalized human neural stem cells were established, their karyotypes were analyzed.
  • Karyotype refers to the phenotype of the chromosome group in metaphase of mitosis, including chromosome number, size, and morphological characteristics.
  • the constructed conditionally immortalized human neural stem cell chromosomes were measured and calculated, followed by grouping, queuing, pairing and morphological analysis.
  • Example 5 The proliferation rate of conditionally immortalized human neural stem cells is accelerated by drug induction
  • the recombinant human neural stem cells were divided into two groups, the control group was added with complete medium of neural stem cells, and the experimental group was added with complete medium of neural stem cells containing 100nM 4-hydroxytamoxifen. After 4 days of culture, PCR was performed to detect the transcription of c-myc downstream genes.
  • the above-mentioned neural stem cells were collected for mRNA extraction, reverse-transcribed into cDNA, and then detected by PCR.
  • the detection index is c-myc downstream activation gene, including cad, mrdb, ord, rccl and rcl and other sequences.
  • conditional immortalized human neural stem cells were adhered to the wall, they were placed in a long-term monitoring device to monitor the dynamic process of cell proliferation.
  • the growth trend of the cells was detected according to the fusion area of the recombinant neural stem cells, and the comparison between adding 4-hydroxytamoxifen and The difference of cell proliferation rate in the experimental group without 4-hydroxytamoxifen.
  • the human neural stem cells to be tested were divided into two groups, respectively (A) recombinant neural stem cells added with complete neural stem cell medium; (B) recombinant neural stem cells added with neural stem complete medium containing 100 nM 4-hydroxytamoxifen. Two groups of neural stem cells were seeded in six-well plates, each group of cells was seeded into three wells, and the seeding density was 5 ⁇ 10 5 /well. After inoculation, monitor for 4.5 days in the long-term monitoring equipment. The proliferation rate of neural stem cells was compared between the two groups according to the cell fusion area rate.
  • EdU is a thymidine analogue, which can replace thymidine nucleotide (T) infiltrating into the DNA molecule being replicated during cell proliferation, and detect DNA replication activity based on the specific reaction between EdU and fluorescent dyes. By detecting EdU The markers can accurately reflect the proliferation of neural stem cells.
  • the human neural stem cells to be detected are divided into four groups, respectively (A) neural stem cells are added with complete medium for neural stem cells; (B) neural stem cells are added with complete medium for neural stem cells containing 100nM 4-hydroxytamoxifen; (C) ) The conditional immortalized human neural stem cells were added to the complete neural stem cell medium; (D) the conditionally immortalized human neural stem cells were added to the neural stem complete medium containing 100 nM 4-hydroxytamoxifen.
  • the four groups of neural stem cells were inoculated into confocal small dishes at a seeding density of 1*10 5 /well, EdU dye (purchased from Millipore, #17-10525) was added after 24 hours, and fluorescent staining was performed after co-incubating for 48 hours.
  • the neural stem cells were fixed with 4% paraformaldehyde for 15 minutes before staining, washed with PBS after fixing, and incubated with 0.05% tritonX-100 for 20 minutes to make the cell membrane permeable. Finally, use the EdU dye corresponding to the fluorescent reagent for staining, and observe under the fluorescence microscope after staining. According to the situation of neural stem cells labeled with EdU dye, the differences in the proliferation rate of neural stem cells in the four groups were compared.
  • Example 6 Preparation of cell membrane nanovesicles derived from conditionally immortalized human neural stem cells
  • conditionally immortalized human neural stem cells were expanded and cultured, the cells were digested with accutase, 1x107 cells were collected by centrifugation and resuspended in 5 mL PBS, and the cell suspension was sequentially passed through 10 ⁇ m, 5 ⁇ m, and 1 ⁇ m using an extruder (Sigma, 610000-1EA). Acetic acid membrane with pore size (Whatman, 110615, 110613, 800319), repeated extrusion 8 to 12 times.
  • Use WB to detect the surface markers of nanovesicles derived from human neural stem cells and conditionally immortalized human neural stem cells take 30 ⁇ g of cell membrane nanovesicle suspension, add 5 ⁇ Loading Buffer to boil water for 5 minutes, and use 10% SDS-PAGE gel Electrophoresis for separation. Transfer to PVDF membrane, block with 5% skimmed milk for 1 h, add exosome marker antibody anti-CD9 (1:1 000 dilution), anti-TSG101 antibody (1:1 000 dilution) and incubate overnight at 4°C; Incubate for 60 min at room temperature in the dark, rinse with 1 ⁇ PBST, develop with ECL and take pictures.
  • Nanoparticle tracking analysis was used to detect the diameter distribution and quantity of the prepared conditional immortalized human neural stem cell-derived cell membrane nanovesicles. Take 1ml of cell membrane nanovesicles and push them into the NTA with a syringe, and at the same time The particle size and number of detected cell membrane nanovesicles.
  • the morphology of the prepared cell membrane nanovesicles was detected by transmission electron microscopy. Take 10 ⁇ L of the above cell membrane nanovesicle suspension and sonicate for 20 minutes, fix with an equal volume of 4% paraformaldehyde at room temperature for 30 minutes, settle on the copper grid, and stain with phosphotungstic acid for 5 minutes Afterwards, excess liquid was removed and dried, and the morphology of cell membrane nanovesicles was collected and observed by TEM.
  • HT22 Well-growing mouse neuron cells
  • control group and hypoxia group were respectively used with DMEM complete medium (BI, 06-1055-57-1ACS)
  • DMEM complete medium BI, 06-1055-57-1ACS
  • the medium was changed with HBSS (GIBCO, 24010043), and placed in a 37°C cell culture incubator (5% CO 2 and 95% N 2 ) for 10 hours.
  • Image-iT TM Green hypoxia monitoring reagent (Thermo, I14833). This reagent is a novel, immobilizable fluorescent compound for the measurement of hypoxia in living cells. Living cells do not fluoresce in environments with normal oxygen concentrations, but do fluoresce when oxygen levels are reduced. Image-iT Green Hypoxia Monitoring Reagent maintains its fluorescence when cells/tissues return to normal oxygen levels.
  • hypoxia monitoring reagent stock solution was diluted in the culture medium with a final concentration of 1 ⁇ m, and the hypoxia group and the control group were incubated at 37° C. for 30 min.
  • Annexin V is a series of calcium ion-dependent phospholipid-binding proteins that can bind to phosphatidylserine (Phosphatidylserine, PS) to identify apoptotic cells. In healthy cells, PS is predominantly located on the cytosolic side of the plasma membrane.
  • Annexin V staining positive (PI negative) are in the early stage of apoptosis.
  • Annexin V staining used in conjunction with PI can be widely used to identify the stage of apoptosis by flow cytometry analysis.
  • the neurons were divided into three groups: the control group (Control), the hypoxia group (OGD) and the conditionally immortalized human neural stem cell-derived cell membrane nanovesicle treatment group (NVs) after hypoxia.
  • Control the control group
  • OGD hypoxia group
  • NVs conditionally immortalized human neural stem cell-derived cell membrane nanovesicle treatment group
  • In the control group neurons were cultured normally; in the hypoxia group, neurons were cultured at 37°C for 10 h in hypoxia equipment (5% CO 2 and 95% N 2 ); Incubate the neurons for 24 hours (the final concentration of vesicles is 20 ⁇ g/ml).
  • the cells in the staining group were resuspended in 5 ⁇ L Annexin V-FITC and 195 ⁇ L Binding Buffer (1x) at a cell density of 2-5 ⁇ 10 5 /mL, and incubated at room temperature for 10 min in the dark.

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Abstract

提供了一种条件永生化人神经干细胞来源细胞膜纳米囊泡制剂及其制备方法和应用，属于细胞工程和基因工程技术领域。通过基因工程技术对人神经干细胞进行改造，使其成为条件永生化细胞，能够在体外进行大批量扩增。随后通过物理挤压技术促使条件永生化人神经干细胞的细胞膜再融合，从而大规模制备出具有母体细胞特征的细胞膜纳米囊泡，构建了一套从细胞到后续细胞衍生物制剂制备的完整系统，可解决人神经干细胞衍生物制剂面向临床转化所遇到的制剂难以大规模生产等问题。通过建立条件永生化人神经干细胞来源细胞膜纳米囊泡制剂的制备方法，为人神经干细胞衍生物制剂应用于临床提供了强有力的基础。

Description

一种条件永生化人神经干细胞来源细胞膜纳米囊泡制剂及其制备方法和应用 技术领域
本发明属于细胞工程和基因工程技术领域，具体涉及一种条件永生化人神经干细胞来源细胞膜纳米囊泡制剂及其制备方法和应用。
背景技术
由于中枢神经系统(central nervous system,CNS)固有的复杂性，CNS相关治疗学的发展仅取得了有限的成功。近年来，干细胞治疗促进了多种难治性中枢神经系统疾病的临床进展，并在世界范围内取得了一定的疗效。尤其是神经干细胞作为中枢神经系统来源的种子细胞，在神经系统疾病的治疗中具有更为有效的作用。大量的临床前研究表明，神经干细胞移植作为一种有潜力的治疗手段，通过产生神经营养因子、减轻神经炎症、增强神经可塑性和细胞替代等多种机制在中枢神经系统疾病中发挥着重要作用。然而，迄今为止只有少数临床研究成果发表，神经干细胞的临床应用面临着前所未有的挑战。其主要障碍是高度复杂的生命过程难以被体外过程控制，移植细胞存活率低，免疫排斥和肿瘤形成的风险，人们对这些细胞是否适合进一步应用的担忧。此外，神经干细胞的特性使移植更加困难，包括增殖速率低、来源有限、伦理问题等。因此，需要开发一种有效的内在生物活性载体，保留功能和活性，同时降低与干细胞治疗相关的风险，如不必要的复制、分化和血管阻塞。
神经干细胞衍生的细胞外小泡(Extravesicles,Evs)为基础治疗提供了一种特别有吸引力的细胞移植替代方案，与细胞治疗不同，EVs给药后安全性问题的概率几乎为零，因为它们不具有核细胞，不能复制，在进入细胞后很快降解。许多研究表明，神经干细胞介导的功能恢复主要归因于神经干细胞旁分泌信号提供的营养支持，包括生长因子和细胞外小泡，如外泌体。干细胞被用作治疗药物的生产者，而不是作为治疗药物本身，这增强了治疗的可行性。因此，无细胞治疗引起了人们极大的兴趣。为了使无细胞产品能够取代细胞疗法成为更安全、更廉价的生物药物进入临床，无细胞疗法的大规模生产成为生产制造业的一大挑战。因此，亟待开发一种高效的、并可以大规模制备的神经干细胞来源的细胞衍生物制剂。
发明内容
鉴于此，本发明的目的是提供一种条件永生化人神经干细胞来源的细胞膜纳米囊泡制剂及其制备方法，构建了一套从细胞到后续细胞衍生物制剂制备的完整系统，用 于解决人神经干细胞衍生物面向临床转化所遇到的难以大规模生产的问题。
本发明目的是通过以下方式实现：
本发明首先通过基因工程技术对人神经干细胞进行改造，构建条件永生化人神经干细胞，加快其增殖速率，使其能够实现大规模培养，保证细胞膜纳米囊泡制剂制备来源的稳定性。
一种条件永生化人神经干细胞来源细胞膜纳米囊泡制剂的制备方法，主要包括以下步骤：
(1)通过基因工程手段构建条件永生化人神经干细胞，所述条件永生化人神经干细胞在特定药物的作用下增殖速度加快，能够进行大批量扩增培养；
(2)向步骤(1)得到的条件永生化人神经干细胞的培养体系中加入所述的特定药物，提高所述条件永生化人神经干细胞的增殖速度，培养后获得大批量的条件永生化人神经干细胞；
(3)通过物理挤压方法促使步骤(2)得到的条件永生化人神经干细胞膜再融合，获取具有母体细胞特征的细胞膜纳米囊泡。
基于上述技术方案，进一步地，步骤(1)中所述永生化人神经干细胞表达c-myc ER系统(雌激素诱导型c-myc系统)。
基于上述技术方案，进一步地，所述c-myc ER系统由c-myc与雌激素受体ER融合而成。
基于上述技术方案，进一步地，所述c-myc ER系统的核苷酸序列如SEQ ID NO:1所示。
基于上述技术方案，进一步地，步骤(1)中所述构建条件永生化人神经干细胞的具体过程为将编码c-myc ER系统的基因直接导入宿主细胞内或者通过病毒转染方式将携带编码c-myc ER系统的基因的重组病毒导入到宿主细胞内。
基于上述技术方案，进一步地，所述条件永生化人神经干细胞表达的c-myc ER系统的雌激素受体的配体结合区域包含点突变(G521R)。
基于上述技术方案，进一步地，所述特定药物包括他莫昔芬、4-羟基他莫昔芬。
基于上述技术方案，进一步地，所述特定药物在培养体系的终浓度为10-1000nM。
基于上述技术方案，进一步地，所述特定药物对永生化细胞的增殖速率调控具有可逆性，当向永生化细胞加入药物后，永生化细胞反应性增殖速率加快，且当停止加药后，永生化细胞的增殖速率会逐渐恢复至原水平。
基于上述技术方案，进一步地，步骤(3)中所述物理挤压方法的具体操作步骤包括将所述条件永生化人神经干细胞的细胞悬液通过挤出器依次通过10μm、5μm、1μm孔径尺寸的滤膜，反复挤出5～20次，收集所得混悬液，于0～4℃，1000～5000g离心5～30min，取上清，于0～4℃，10000～30000g离心5～30min，弃上清，所得沉淀用缓冲液重悬得到细胞膜纳米囊泡。
本发明另一方面提供一种细胞膜纳米囊泡，所述细胞膜纳米囊泡是由上述制备方法制得。
本发明另一方面提供上述的细胞膜纳米囊泡在制备治疗中枢神经系统疾病的药物中的应用。
本发明相对于现有技术具有的有益效果如下：
1.本发明的可大规模制备的条件永生化人神经干细胞来源细胞膜纳米囊泡制剂及其制备方法是本发明人首先开发并提出的，通过基因工程技术对人神经干细胞进行条件永生化改造，使其能够在体外进行大批量扩增培养，从而为人神经干细胞衍生物制剂提供稳定的制备来源。随后通过物理挤压促使细胞膜再融合技术大规模制备具有母体细胞特征的来源于条件永生化人神经干细胞的细胞膜纳米囊泡。构建了一套从细胞到后续细胞衍生物制剂制备的完整系统，可解决人神经干细胞衍生物制剂面向临床转化所遇到的制剂难以大规模生产的问题。
2.本发明的可大规模制备的条件永生化人神经干细胞来源细胞膜纳米囊泡制剂的制备方法具有产率高，操作简单，可重复性强等特点。同时在对人神经干细胞进行基因工程修饰时所选取的条件永生化构建方案，可通过外源添加药物对其增殖速率进行调控，保证了制剂来源母体细胞的安全性。
3.本发明通过建立条件永生化人神经干细胞来源细胞膜纳米囊泡制剂的制备方法，提供了一整套从细胞到细胞衍生物制剂制备的完整方案，为后续其他细胞来源的细胞衍生物制剂面向临床转化研究提供参考。
附图说明
为了更清楚地说明本发明实施例，下面将对实施例涉及的附图进行简单地介绍。
图1为本发明的条件永生化人神经干细胞来源细胞膜纳米囊泡制剂制备方法流程图；
图2为本发明实施例1中不同代次的人神经干细胞(第2代、4代和8代)的光 学显微镜图片；
图3为本发明实施例1中的人神经干细胞的特异性标志物鉴定图片；
图4为本发明实施例1中的人神经干细胞诱导分化能力检测图片；
图5为本发明实施例1中编码雌激素诱导型c-myc系统的慢病毒表达载体图谱；
图6为本发明实施例1中慢病毒感染后人神经干细胞在荧光显微镜下的图片；
图7为本发明的条件永生化人神经干细胞核型分析图片；
图8为本发明实施例1中的重组人神经干细胞加入4-羟基他莫昔芬后下游基因转录图片，其中，4-OHT为加药组，un-4-OHT为完全培养基实验组；
图9为本发明实施例1中的重组人神经干细胞未加或加入4-羟基他莫昔芬后增殖速率变化图片，其中，4-OHT为加药组，un-4-OHT为完全培养基实验组；
图10为本发明的人神经干细胞和重组人神经干细胞EdU标记检测增殖实验；
图11为本发明的实施例6中条件永生化人神经干细胞来源细胞膜纳米囊泡Western Blot图；
图12为本发明的实施例6中条件永生化人神经干细胞来源细胞膜纳米囊泡透射电镜图；
图13为本发明的实施例6中条件永生化人神经干细胞来源细胞膜纳米囊泡粒径分布图；
图14为本发明的实施例7中神经元缺氧损伤模型的构建；
图15为本发明的实施例7中永生化人神经干细胞来源细胞膜纳米囊泡治疗后的凋亡情况。
具体实施方式
下面结合实施例对本发明进行详细的说明，但本发明的实施方式不限于此，显而易见地，下面描述中的实施例仅是本发明的部分实施例，对于本领域技术人员来讲，在不付出创造性劳动性的前提下，获得其他的类似的实施例均落入本发明的保护范围。
下述实施例中，如无特殊说明，所使用的实验方法均为常规方法，所用材料、试剂等均可从生物或化学试剂公司购买。
实施例1：人神经干细胞的培养及鉴定
本发明通过对人神经干细胞(NSC)进行改造，从而促进其增殖能力。首先，对人神经干细胞进行培养及鉴定。
人神经干细胞的培养方式为在神经干细胞完全培养基(购买于Stem cell  technology，catalog#05751)中悬浮培养。具体操作方式为：
在生物安全柜中进行神经干细胞原代的提取制备，操作过程需严格无菌操作。取直径100mm无菌培养皿，倒入10mL无菌生理盐水，将完整的流产的胎儿组织(符合《人胚胎干细胞研究伦理指导原则》)置于生理盐水溶液中进行反复冲洗，直至生理盐水澄清透明。使用灭菌手术剪及镊子剥离流产胎儿头部的皮肤，剪开颅骨充分暴露脑组织，用手术镊轻轻撕去脑组织周围软脑膜，尽量剥离血管。将胎脑组织完整的取出，放入新的盛有无菌生理盐水的培养皿中，仔细分离脑皮层。准备35mm无菌培养皿，放入神经干完全培养基，将分离出的皮层组织放入培养基中并分割成1mm大小的组织块。将含有组织块的完全培养基用移液器转移至50mL离心管中，轻柔吹打30次，放入37℃孵箱，静置10分钟，取上清放入新的50mL离心管，300g离心5分钟，弃上清。向50mL离心管中加入2ml Accutase(购买于Stem cell technology，catalog#07920)轻轻吹打混匀，将离心管放入37℃、5％CO 2孵箱消化1分钟，室温下300g离心5分钟，弃上清。向离心管中加入1mL NSC完全培养基，轻柔吹打至形成单细胞悬液并进行细胞计数。取T75细胞培养瓶，加入20mL完全培养基，将计数后的细胞以2x10 4～5x10 4个/mL的密度接种到培养瓶中，标记好细胞批次编号、细胞代数、操作者姓名、操作时间。接种后放于普通光学显微镜下镜检观察，确定细胞在每个培养容器中大致分布均匀，如果细胞分布不均匀，则重新晃动容器。记录细胞状态和异常情况。镜检后的细胞放入二氧化碳培养箱中，培养温度为37℃、CO 2浓度为5％。细胞接种后2-3天，镜下观察，记录细胞生长状态，并进行补液，补液量为2-3mL。之后每3天进行细胞换液，提取后进行原代细胞悬浮培养，当神经球生长至100-150μm时进行神经干细胞传代。
将含有神经球的培养液转移到15或50mL的离心管中，室温下，200g离心2分钟，离心后弃掉大部分的上清液，使神经球留在较小体积的培养基中，加入1mL Accutase酶，37℃孵育5min，间断轻轻吹打，以确保细胞不会聚集或沉淀在试管底部。使用1000μL枪头轻柔吹打神经球，形成单细胞悬浮液，加入4mL完全培养基终止消化。室温下，300g离心3min，弃上清液，用新鲜培养基重悬细胞，将细胞稀释至2～5x10 4个/mL的密度接种到培养瓶中，放入37℃，5％CO 2的孵箱继续培养。
通过光学显微镜观察不同代次的人神经干细胞的细胞形态，结果如图2所示，可以看出，不同代次的细胞的生长状态良好，不同代次之间没有明显的差异，进一步证明提取的人神经干细胞在体外培养的稳定性。
基于人神经干细胞高表达Nestin和Sox2，通过流式细胞仪对本发明的人神经干细胞进行特异性标志物的鉴定，具体操作步骤为：用Accutase酶消化细胞，加入神经干细胞完全培养基将神经干细胞吹打至单细胞，将获得的神经干细胞用4％多聚甲醛进行破膜固定15min后，300g离心5min，弃上清，再加入2mL 1×PBS，充分混匀，再次300g离心5min，弃上清，加入1×PBS重悬后制备单细胞悬液(细胞密度为1x10 6/mL)。取100μl加入标记好的流式管中，分别与目的抗体(Nestin抗体、Sox2抗体)避光共孵育30min。孵育结束后，在各管中加入1ml 1×PBS，充分混匀，300g离心5分钟，弃上清液，洗涤二次后，上机选择相应抗体对应波长进行检测。
人神经干细胞特异性标志物的鉴定结果如图3所示，结果表明，本发明所培养的人神经干细胞高表达Nestin和Sox2，未出现分化，建系前的人神经干细胞为正常的神经干细胞。
基于人神经干细胞具有分化为神经元、星形胶质细胞和少突胶质细胞的能力，因此，对本发明的人神经干细胞进行分化能力的检测。具体操作步骤为：将神经干细胞按照4×10 5个细胞/孔在matrigel包被的六孔板内培养，并将神经干细胞增殖培养基替换为神经干细胞分化培养基(购买于Stem cell technology，#05752)，每孔加入2ml。每隔2～3天更换一次分化培养基，于分化诱导培养10天后，于CQ1激光共聚焦高内涵细胞分筛系统(YOKOGAWA,CQ1)进行免疫荧光染色鉴定，验证提取的人神经干细胞具有向神经系统三大神经细胞分化的能力。
人神经干细胞诱导分化能力检测结果如图4所示，A：神经干细胞诱导分化第2天及第4天普通光学显微镜下观察的细胞形态；B：神经元免疫荧光染色：小鼠单克隆抗体beta III Tubulin(Tuj1)及DAPI染色；C：少突胶质细胞免疫荧光染色：兔多克隆抗体Oligodendrocyte Specific Protein及DAPI染色；D：星型胶质细胞免疫荧光染色：兔多克隆抗体GFAP及DAPI染色；进一步证明本发明提取的人神经干细胞具有向神经系统三大神经细胞分化的能力。
实施例2：表达载体的构建
本实施例通过慢病毒感染宿主细胞的方法来改造细胞。因此，首先构建包含雌激素诱导型c-myc系统的表达载体，将c-myc ER基因构建于慢病毒载体中，使用的慢病毒表达载体为GV492(购买于上海吉凯基因化学技术有限公司)；其中，病毒表达载体GV492图谱如图5所示，c-myc ER基因序列如SEQ ID NO:1所示。
具体步骤如下：
1.目的基因的扩增
(1)载体酶切：配制50μl酶切体系。加入混合试剂，用移液器轻轻吹打混匀，短暂离心，置于37℃反应3h或过夜。对载体酶切产物进行琼脂糖凝胶电泳，回收目的条带。
(2)目的基因片段的获取：人工合成c-myc ER基因序列，配制反应体系，轻轻吹打混匀，短暂离心，置于PCR仪中进行反应。
(3)PCR产物与载体连接：配制反应体系，用移液器轻轻吹打混匀，短暂离心，避免产生气泡，于37℃反应30min，随后置于冰水浴中冷却5min后立即转化。
(4)转化：将10μL连接反应产物加入到100μL感受态细胞中，轻弹管壁数下混匀，在冰上放置30min，42℃热激90s，冰水浴孵育2min，加入500μL LB培养基，置于37℃摇床振荡培养1h。取适量菌液均匀涂布在含有相应抗生素的平板上，在恒温培养箱中倒置培养12-16h。
(5)菌落PCR鉴定：配制鉴定体系，震荡混匀，短暂离心。在超净工作台中，用无菌的枪头挑取单个菌落至20μL鉴定体系中，吹打混匀，置于PCR仪中进行反应。
(6)测序：将鉴定出的阳性克隆转化子接种于适量含相应抗生素的LB液体培养基中，37℃培养12-16h，取适量菌液进行测序。对测序结果与目的基因序列进行比对分析。比对结果说明：测序结果与目标序列完全一致。
2.质粒抽提
将测序正确的菌液转接于10ml含相应抗生素的LB液体培养基中，37℃培养过夜，用天根无内毒素质粒小提中量试剂盒进行质粒抽提，抽提合格的质粒进入下游流程。详细操作步骤如下：
(1)收集过夜培养的菌液于标记好的5ml离心管，12000rpm，离心2min收菌；
(2)弃上清，加入250μl细胞重悬液，充分振荡，使菌块悬浮均匀；
(3)加入250μl细胞裂解液，再加入10μl蛋白酶K，上下颠倒5-6次，轻轻混匀；静置1-2min，致使菌体裂解澄清；
(4)加入350μl中和液，上下颠倒混匀，使蛋白完全析出，冰浴静置5min；
(5)10000rpm离心10min，弃蛋白，收集上清于另一干净无菌的1.5ml EP管；
(6)12000rpm离心5min，同时准备标记好的回收柱，将上清转至回收柱中，12000rpm离心1min，弃下层废液；
(7)加入600μl预先配置好的漂洗液，12000rpm离心1min，弃下层废液，重复一次，12000rpm空离2min，进一步除去残留的漂洗液；
(8)在超净台中将回收柱转移至新的1.5ml EP管中，静置10-20min，自然晾干；
(9)往回收柱中加入95μl Nuclease-Free Water，静置2min，12000rpm离心2min，收集样品做好编号，电泳、测定浓度，进行质检。
实施例3：质粒转染与慢病毒收获
采用质粒共转染293T细胞。在转染完成后的48-72h进行病毒收获(即未纯化的细胞上清液)，根据不同的实验需求，确定采用相应的浓缩纯化方式得到高滴度的慢病毒保存液，最后根据严格的质量标准测定慢病毒的各项指标。在一定滴度范围内的慢病毒颗粒可以满足大部分体内外实验需求，流程如下。
1.质粒转染
(1)转染前24h，用胰蛋白酶消化对数生长期的293T细胞，以含10％血清的培养基调整细胞密度约5x 10 6细胞/15ml，重新接种于10cm细胞培养皿，37℃、5％CO 2培养箱内培养。24h待细胞密度达70％～80％时即可用于转染；
(2)转染前2h更换为无血清培养基；
(3)向一支灭菌离心管中加入所制备的各DNA溶液(GV492载体质粒20μg、pHelper1.0载体质粒15μg、pHelper 2.0载体质粒10μg)，与相应体积的Fugene6转染试剂(Boehringer Mannheim)混合均匀，调整总体积为1ml，在室温下温育15min；
(4)混合液缓慢滴加至293T细胞的培养液中，加入过程一定要均匀，尽可能地不要将细胞吹起，混匀，于37℃、5％CO 2细胞培养箱中培养；
(5)培养6h后弃去含有转染混合物的培养基，加入10ml的PBS液清洗一次，轻柔晃动培养皿以洗涤残余的转染混合物后倒弃；
(6)缓慢加入含10％血清的细胞培养基20ml，于37℃、含5％CO 2培养箱内继续培养48-72h。
2.慢病毒浓缩与纯化
(1)根据细胞状态，收集转染后48h(转染即0h计起)的293T细胞上清液；
(2)于4℃，4000g离心10min，除去细胞碎片；
(3)以0.45μm滤器过滤上清液于40ml超速离心管中；
(4)分别配平样品，将带有病毒上清液的超速离心管逐一放入至Beckman超 速离心机内,设置离心参数为25000rpm，离心时间为2h，离心温度控制在4℃；
(5)离心结束后，弃去上清，尽量去除残留在管壁上的液体，加入病毒保存液(可用PBS或细胞培养基替代)，轻轻反复吹打重悬；
(6)经充分溶解后，高速离心10000rpm，离心5min后，取上清按要求分装；
(7)准备样品待检测。
3.慢病毒质量检测
慢病毒的质量控制要点包括物理状态检测、无菌检测及病毒滴度检测。
(1)物理指标检测
1)颜色判定：通过肉眼判定，慢病毒保存液呈粉红色澄清液体状；
2)粘稠度判定：用20-200μl规格移液器缓慢吸取50μl慢病毒保存液体，无明显粘稠感或吸液滞后现象；
(2)无菌检测：将病毒加入293T细胞验证，正常培养24h后镜检，无任何细菌及真菌污染情况，同时参照空细胞组，细胞间隙无明显颗粒存在，培养基澄清透明。
(3)滴度检测及分析：绝对定量qPCR法
慢病毒可以将病毒的5’LTR—3’LTR区整合入宿主基因组中稳定表达，通过病毒感染工具细胞293T，用绝对定量方法检测出工具细胞293T基因组中的病毒特征单拷贝基因A和宿主特征单拷贝基因B。计算出每个细胞中平均感染病毒颗粒数，再乘以每孔的细胞个数，除以感染量，即可得出病毒样品的滴度。
计算公式：
qPCR滴度(TU/ml)＝N*C/V
N＝感染时24孔板中对应孔的细胞数量；
C(每个细胞中含有的慢病毒个数)＝(A拷贝数/B拷贝数)*2；
V＝对应孔中感染的慢病毒体积(ml)。
实验步骤：
1)标准品制备：构建含有慢病毒基因组保守序列a的质粒标准品A和含有工具细胞293T基因组内单拷贝基因b的质粒标准品B；浓度1×10 10copy/μl，分装后-80℃长久保存。
2)引物设计和制备：分别设计针对质粒标准品A和B设计qPCR引物，并配置成10μM的引物工作液。
3)样品制备：
a)感染前24h，在24孔板中培养293T细胞，密度为5×10 4cell/孔。
b)感染时，收集2-3孔空白对照细胞，分别计数每孔内感染时细胞总数N，每孔感染病毒体积V ml，每个病毒感染3个复孔。
c)感染后24h，每孔加入1000μl完全培养基，小心操作，不要吹起细胞。
d)感染后72h，吸去上清，荧光拍照；同时分别收集孔内细胞。
e)用天根《细胞、血液基因组提取试剂盒》提取收集细胞的基因组。
4)标准品稀释：
10倍梯度稀释法，以10 9、10 8、10 7、10 6、10 5、10 4、10 3、10 2、10 1梯度稀释质量标准品A和B，以及待测样品。
5)配制PCR反应体系。
6)PCR反应：
设定程序为两步法Real-Time定量。预变性95℃，15s，之后每一步变性95℃，5s，退火延伸60℃，30s，共进行40个循环。每次在延伸阶段读取吸光值。PCR结束后，制作溶解曲线，在95℃变性1min，然后冷却至60℃，1min，使DNA双链充分结合。从60℃开始，每步增加0.5℃，保持30s，同时读取吸光值。
7)滴度结果
根据计算公式：qPCR滴度(TU/ml)＝N*C/V，计算样品的平均滴度。
实施例4：条件永生化人神经干细胞的构建
使用神经干细胞完全培养基培养人源神经干细胞，然后以感染复数(MOI)＝5的病毒滴度添加上述方法制得的慢病毒感染人源神经干细胞，从而构建得到过表达c-myc ER的重组人源神经干细胞。由于病毒载体具有GFP标签及抗性，经过含有puromycin的抗性培养基筛选高表达的重组人源神经干细胞。随后通过荧光显微镜下细胞情况与普通光镜下细胞情况进行对比从而判断感染效率。
具体步骤如下：
(1)使用完全培养基基制备密度为1×10 5个/ml人源神经干细胞悬液，以4×10 4个/ml接种至六孔板的每个孔中。
(2)待细胞贴壁后，对细胞进行换液，根据细胞MOI＝5加入相应数量c-myc ER慢病毒，37℃培养12-16h，更换完全培养基继续培养。
(3)感染后约72小时，观察感染效率。感染效率80％为最佳的感染效率。
本发明重组人源神经干细胞感染情况如图6所示，A：重组人源神经干细胞在普 通光学显微镜下的图片；B：重组人源神经干细胞在荧光显微镜下的图片。由此可见，c-myc ER基因在重组人源神经干细胞中成功表达。
建立条件永生化人神经干细胞后，对其细胞核型进行分析。核型是指染色体组在有丝分裂中期的表型，包括染色体数目、大小、形态特征等。对构建的条件永生化人神经干细胞染色体进行测量计算，随后进行分组、排队、配对并进行形态分析。
结果如图7所示，构建的条件永生化人神经干细胞染色体核型正常。
实施例5：通过药物诱导条件永生化人神经干细胞增殖速率加快
1.通过下游基因表达情况验证药物可诱导c-myc蛋白激活
通过向重组神经干细胞内加入4-羟基他莫昔芬，激活细胞内c-myc蛋白活性，通过PCR的方法检测其下游基因的转录情况，下游基因的选择包括cad、mrdb、ord、rccl和rcl。
具体步骤如下：
(1)配置包含100nM 4-羟基他莫昔芬的神经干细胞完全培养基；
(2)将重组人神经干细胞分为两组，对照组加入神经干细胞完全培养基，实验组加入含100nM 4-羟基他莫昔芬的神经干完全培养基。培养4天后，进行PCR检测c-myc下游基因转录情况。
(3)分别取上述神经干细胞进行mRNA提取，逆转录为cDNA后进行PCR检测。检测指标为c-myc下游激活基因，包括cad、mrdb、ord、rccl和rcl等序列。
加入4-羟基他莫昔芬的重组神经干细胞与未加4-羟基他莫昔芬的重组神经干细胞下游基因转录情况对比如图8所示，由此可见，加入4-羟基他莫昔芬的重组神经干细胞(左侧)的cad、mrdb、ord、rccl和rcl的表达量均明显高于未加4-羟基他莫昔芬(右侧)的重组神经干细胞实验组。
2.通过细胞的融合面积进行评价
待条件永生化人神经干细胞贴壁培养后放于长时程监测设备监测细胞增殖动态过程，根据重组神经干细胞的贴壁的融合面积检测细胞的增长趋势，比较加入4-羟基他莫昔芬与未加4-羟基他莫昔芬实验组细胞增殖速率的差异。
具体步骤如下：
将待检测的人神经干细胞分为两组，分别为(A)重组神经干细胞加入神经干细胞完全培养基；(B)重组神经干细胞加入包含100nM 4-羟基他莫昔芬的神经干完全培养基。将两组神经干细胞分别接种于六孔板内，每组细胞接种三个孔，接种密度为 5×10 5/孔。接种好后，在长时程监测设备中监测4.5天。根据细胞的融合面积速率比较两组神经干细胞的增殖速率。
加入4-羟基他莫昔芬的重组神经干细胞与未加4-羟基他莫昔芬的条件永生化人神经干细胞的增殖效率对比如图9所示，进一步证明了加入4-羟基他莫昔芬的条件永生化人神经干细胞与未加4-羟基他莫昔芬的条件永生化人神经干细胞增殖效率存在显著差异，加入4-羟基他莫昔芬的条件永生化人神经干细胞具有更快的增殖速率。
3.通过EdU标记检测神经干细胞增殖情况
EdU是一种胸腺嘧啶核苷类似物，能够在细胞增殖时期代替胸腺嘧啶核苷酸(T)渗入正在复制的DNA分子，通过基于EdU与荧光染料的特异性反应检测DNA复制活性，通过检测EdU标记便能准确地反映神经干细胞的增殖情况。
具体步骤如下：
将待检测的人神经干细胞分为四组，分别为(A)神经干细胞加入神经干细胞完全培养基；(B)神经干细胞加入包含100nM 4-羟基他莫昔芬的神经干完全培养基；(C)条件永生化人神经干细胞加入神经干细胞全培养基；(D)条件永生化人神经干细胞加入包含100nM 4-羟基他莫昔芬的神经干完全培养基。将四组神经干细胞接种于共聚焦小皿内，接种密度为1*10 5/孔，24h后加入EdU染料(购买于Millipore，#17-10525)，共孵育48h后进行荧光染色。
染色前用4％多聚甲醛将神经干细胞进行固定15min，固定后PBS进行清洗，清洗后使用0.05％tritonX-100对细胞孵育20min，使其细胞膜变通透。最后，使用EdU染料对应荧光试剂进行染色，染色结束后在荧光显微经下观察。根据EdU染料标记神经干细胞的情况，比较四组神经干细胞增殖速率的差异。
四组神经干细胞的增殖速率对比如图10所示，由此可见，加入4-羟基他莫昔芬的条件永生化人神经干细胞与其他三组神经干细胞增殖效率存在显著差异，其增殖效率最快，而其他三组神经干细胞增殖速率未见明显差异。
实施例6:制备来源于条件永生化人神经干细胞的细胞膜纳米囊泡
1.条件永生化神经干细胞来源细胞膜纳米囊泡的制备
将条件永生化人神经干细胞进行扩大培养，用accutase消化细胞，离心收集1x10 7个细胞用5mL PBS重悬，利用挤出器(Sigma，610000-1EA)将细胞悬液依次通过10μm、5μm、1μm孔径尺寸的醋酸膜(Whatman，110615，110613，800319)，反复挤出8～12次。收集挤膜完成的混悬液，于4℃，3000g离心15min去除细胞碎片，取上清， 于4℃，20000g离心15min，弃上清，沉淀用200-500μL PBS重悬得到条件永生化人神经干细胞膜来源细胞膜纳米囊泡；全程置于冰上或者低温环境中，防止蛋白变性。
2.神经干细胞纳米囊泡的表面标志物表征
利用WB对人神经干细胞与条件永生化人神经干细胞来源的纳米囊泡表面标志物进行检测，取30μg细胞膜纳米囊泡悬液，加入5×Loadding Buffer沸水中处理5min，用10％SDS-PAGE胶电泳进行分离。转印至PVDF膜上，5％脱脂牛奶封闭1h，分别加外泌体标志分子抗体anti-CD9(1∶1 000稀释)、anti-TSG101抗体(1∶1 000稀释)4℃孵育过夜；二抗室温避光孵育60min，1×PBST漂洗后ECL显影拍照。
结果如图11所示，可以看出人神经干细胞与条件永生化人神经干细胞来源细胞膜纳米囊泡表面标志物CD9与TSG101均为阳性。
3.神经干细胞纳米囊泡的粒径、数量表征
利用纳米颗粒跟踪分析(nanopaticle tracking analysis，NTA)对制备的条件永生化人神经干细胞来源细胞膜纳米囊泡的直径分布和数量进行检测，取1ml细胞膜纳米囊泡，使用针筒注射器推入NTA，同时检测的细胞膜纳米囊泡的粒径和数量。
结果如图12所示，可以看出，条件永生化人神经干细胞来源细胞膜纳米囊泡的平均粒径为182.4nm。经计算，1.2×10 7个细胞制备出2.61×10 11个细胞膜纳米囊泡，单个细胞能够制备21750个细胞膜纳米囊泡，表明本发明制备细胞膜纳米囊泡的方法产率高，适宜工业化生产。
4.条件永生化人神经干细胞膜纳米囊泡的形态学表征
利用透射电镜对制备的细胞膜纳米囊泡进行形态学检测，取上述细胞膜纳米囊泡悬液10μL超声20min，用等体积4％多聚甲醛室温固定30min，沉降在铜网上，用磷钨酸染色5min后，除去多余的液体并干燥，通过TEM采集并观察细胞膜纳米囊泡的形貌。
结果如图13所示，观察到细胞膜纳米囊泡均为脂质双分子层结构，呈圆形或杯口形。
实施例7:条件永生化人神经干细胞来源细胞膜纳米囊泡修复神经元损伤
1.神经元缺氧损伤模型的构建
选用生长良好的小鼠神经元细胞(HT22),分为对照组与缺氧组，传代贴壁后，对照组与缺氧组分别使用DMEM完全培养基(BI,06-1055-57-1ACS)与HBSS(GIBCO，24010043)换液,置于37℃细胞培养箱(5％CO 2和95％N 2)培养10小时。
2.神经元缺氧损伤模型的检测
使用Image-iT TM Green缺氧监测试剂(Thermo，I14833)进行神经元缺氧模型的检测。该试剂是一种新型、可固定的荧光化合物，用于测定活细胞中的缺氧情况。活细胞在具有正常氧气浓度的环境中不发荧光，而当氧水平降低时会发出荧光。当细胞/组织恢复至正常氧水平时，Image-iT Green缺氧监测试剂可维持其荧光。
具体步骤如下：
(1)将2×10 5个/孔HT22细胞铺于22mm共聚焦小皿中过夜。
(2)将缺氧监测试剂储备溶液以1μm最终浓度的稀释于培养基，缺氧组与对照组共同在37℃下孵育30min。
(3)将对照组细胞放置于正常培养条件下(5％CO 2)37℃培养10h，将缺氧组放入缺氧设备中(5％CO 2和95％N 2)37℃培养10h。
(4)10小时后使用荧光显微镜成像。
结果如图14所示，由对照组(Control)与缺氧组(OGD)成像效果图可知缺氧模型构建成功。
3.条件永生化人神经干细胞来源细胞膜纳米囊泡治疗缺氧损伤神经元
使用eBioscience TM Annexin V-FITC Apoptosis Detection Kit(Thermo，BMS500FI-100)检测囊泡治疗缺氧损伤神经元效果。膜联蛋白V(Annexin V)是一系列钙离子依赖型磷脂结合蛋白，可结合至磷脂酰丝氨酸(Phosphatidylserine,PS)以鉴定凋亡细胞。在健康细胞中，PS主要位于质膜的细胞溶质一侧。细胞凋亡开始后，PS在磷脂双分子层中的非对称分布逐渐消失，并转移至细胞外膜中，这一点可通过荧光标记的Annexin V进行检测。在细胞凋亡早期，质膜可阻挡碘化丙啶(Propidium Iodide,PI)等活性染料进入细胞，因此仅显示出Annexin V染色阳性(PI阴性)的细胞处于细胞凋亡早期。在细胞凋亡晚期，由于细胞膜丧失完整性，使得Annexin V结合至细胞溶质PS，并且细胞开始吸收PI。Annexin V染色与PI配合使用，可广泛应用于通过流式细胞仪分析鉴定细胞凋亡阶段。
(1)将神经元分为三组：对照组(Control)、缺氧组(OGD)及缺氧后条件永生化人神经干细胞来源细胞膜纳米囊泡治疗组(NVs)。对照组为正常培养神经元，缺氧组为在缺氧设备中(5％CO 2和95％N 2)37℃培养10h神经元，缺氧后囊泡治疗组为缺氧后与囊泡共孵育(囊泡终浓度为20μg/ml)24h神经元。
(2)将对照组、缺氧组及缺氧后囊泡治疗组神经元在检测前分别用PBS清洗一 遍。
(3)空白组细胞使用200μl Binding Buffer(1x)重悬，细胞密度为2-5×10 5/mL。
(4)染色组细胞使用5μL Annexin V-FITC与195μL Binding Buffer(1x)重悬，细胞密度为2-5×10 5/mL，室温下避光孵育10min。
(5)孵育结束后，向细胞悬液中加入10μl PI(20μg/ml)。
(6)通过流式细胞术进行凋亡分析。
三组神经元流式凋亡分析结果如图15所示，可以看出，相较于缺氧组，细胞膜纳米囊泡治疗组神经元早期凋亡明显减少(Annexin-V阳性，PI阴性)。
最后应说明的是：以上各实施例仅用以说明本发明的技术方案，而非对其限制；尽管参照前述各实施例对本发明进行了详细的说明，本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分或者全部技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本发明各实施例技术方案的范围。
Figure PCTCN2021116164-appb-000001
Figure PCTCN2021116164-appb-000002

Claims (10)

  1. 一种条件永生化人神经干细胞来源细胞膜纳米囊泡的制备方法，其特征在于，包括以下步骤：
    (1)通过基因工程手段构建条件永生化人神经干细胞，所述条件永生化人神经干细胞在特定药物的作用下增殖速度加快；
    (2)向步骤(1)得到的条件永生化人神经干细胞的培养体系中加入所述的特定药物，提高所述条件永生化人神经干细胞的增殖速度，培养获得大批量的条件永生化人神经干细胞；
    (3)通过物理挤压方法促使步骤(2)得到的条件永生化人神经干细胞膜再融合，大规模获取具有母体细胞特征的细胞膜纳米囊泡。
  2. 根据权利要求1所述的制备方法，其特征在于，步骤(1)中所述条件永生化人神经干细胞表达雌激素诱导型c-myc系统。
  3. 根据权利要求2所述的制备方法，其特征在于，所述雌激素诱导型c-myc系统由c-myc与雌激素受体ER融合而成。
  4. 根据权利要求2所述的制备方法，其特征在于，所述雌激素诱导型c-myc系统的核苷酸序列如SEQ ID NO:1所示。
  5. 根据权利要求2-4任一项所述的制备方法，其特征在于，步骤(1)中所述构建条件永生化人神经干细胞的具体过程为将编码雌激素诱导型c-myc系统的基因直接导入宿主细胞内或者通过病毒转染方式将携带编码雌激素诱导型c-myc系统的基因的重组病毒导入到宿主细胞内。
  6. 根据权利要求1所述的制备方法，其特征在于，所述特定药物包括他莫昔芬、4-羟基他莫昔芬；所述特定药物对永生化人神经干细胞的增殖速率的调控具有可逆性。
  7. 根据权利要求6所述的制备方法，其特征在于，所述特定药物在培养体系的终浓度为10-1000nM。
  8. 根据权利要求1所述的制备方法，其特征在于，步骤(3)中所述物理挤压方法的具体操作步骤包括将所述条件永生化人神经干细胞的细胞悬液通过挤出器依次通过10μm、5μm、1μm孔径尺寸的滤膜，反复挤出5～20次，收集所得混悬液，于0～4℃，1000～5000g离心5～30min，取上清，于0～4℃，10000～30000g离心5～30min，弃上清，所得沉淀用缓冲液重悬得到细胞膜纳米囊泡。
  9. 一种细胞膜纳米囊泡，其特征在于，由权利要求1-8任一项所述的制备方法制得。
  10. 权利要求9所述的细胞膜纳米囊泡在制备治疗中枢神经系统疾病的药物中的应用。
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