WO2004038011A1 - Cellules souches neuronales humaines transduites par le gene htert, methode de production et d'identification de ces dernieres - Google Patents

Cellules souches neuronales humaines transduites par le gene htert, methode de production et d'identification de ces dernieres Download PDF

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WO2004038011A1
WO2004038011A1 PCT/CN2002/000745 CN0200745W WO2004038011A1 WO 2004038011 A1 WO2004038011 A1 WO 2004038011A1 CN 0200745 W CN0200745 W CN 0200745W WO 2004038011 A1 WO2004038011 A1 WO 2004038011A1
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neural stem
stem cell
htert
cells
cell line
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PCT/CN2002/000745
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Lingsong Li
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Sinocells Bio Technologies Co., Ltd
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Publication of WO2004038011A1 publication Critical patent/WO2004038011A1/fr

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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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0618—Cells of the nervous system
    • C12N5/0623—Stem cells
    • 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
    • C12N2503/00—Use of cells in diagnostics
    • C12N2503/02—Drug screening
    • 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

Definitions

  • the invention relates to a neural stem cell line, a lineage and identification method thereof.
  • the neural stem cell line can be used as a cell platform for basic research and a cell platform for drug screening for the nervous system. It also has important clinical application value and can be used as a cell carrier for genetically modified genes for neurological diseases (neurological tumors, hereditary metabolic disorders of the nervous system). Neural system degeneration diseases (Parkinson's disease, Alzheimer's disease, lateral sclerosis of the spinal cord), and injury (spinal trauma, pediatric cerebral palsy, cerebrovascular accident) cell transplantation materials for repair, so the establishment of neural stem cell lines has an important basis Research and clinical application value. Background technique
  • the neural stem cell line established by this method is transduced with viral oncogenes, and the protein encoded by it can destroy multiple cell pathways, causing the cell line to lose partial differentiation characteristics, lose normal cell cycle checkpoint control, and increase chromosome instability. It may lead to malignant transformation of cells and make cells have carcinogenic potential (Thomas R Yeager et al., Current Opinion in Biotechnology, 10: 465-469, 1999). This deficiency also limits the clinical application of this cell line.
  • the primary cultured human neural stem cells in vitro under the stimulation of various mitogenic factors (such as: epidermal growth factor EGF, basic fibroblast growth factor bFGF, and leukemia inhibitory factor LIF), maintain a non-differentiated proliferation state ( Me lis sa K. Carpenter et al., Exper imental l Neurology 158: 265-278, 1999).
  • mitogenic factors such as: epidermal growth factor EGF, basic fibroblast growth factor bFGF, and leukemia inhibitory factor LIF
  • telomerase activation is an important factor in cell immortalization.
  • Human telomerase has three subunits, of which the reverse transcriptase catalytic subunit is referred to as hTERT (ie, human Telomerase Reverse Transcriptase).
  • hTERT ie, human Telomerase Reverse Transcriptase
  • Cells surpass the first lethal Ml and Survival mechanisms involve mutations in p53 tumor suppressor genes, pRB mutations, and activation of oncogenes (such as H-ras, HPV16 E6 / E7, adenovirus E1A / E1B, and SV40 large T antigen).
  • telomere activation Cells surpass the second lethal M2 and immortality is closely related to telomerase activation (Colgin LM et al., Curr Opin Genet Dev, 9: 97-103, 1999).
  • the expression of the exogenous telomerase catalytic subunit hTERT gene in normal somatic cells is sufficient to induce telomerase activity, stabilize the telomere length, and enhance cell proliferation ability, thereby greatly extending the life of its in vitro culture and becoming immortalized cells (Vaziri H et al., Curr Biol, 8: 279-282, 1998).
  • the present invention provides a human neural stem cell line transfected with the hTERT gene.
  • the present invention provides a method for establishing a human neural stem cell line transfected with the hTERT gene, comprising: (1) isolating and purifying human neural stem cells; (2) packaging a viral recombinant plasmid carrying the hTERT gene; (3) carrying the hTERT gene The recombinant virus infected neural stem cells; and (4) the neural stem cells were transduced and expanded after transduction of the hTERT gene, thereby obtaining neural stem cell lines transfected with the hTERT gene RT gene.
  • the invention also provides a method for identifying a hTERT gene-transformed neural stem cell line, including identifying the expression of hTERT gene in a cell, the hTERT gene-transformed neural stem cell line in vitro and in vivo proliferation and differentiation ability, and a hTERT gene-transformed neural stem cell.
  • the characteristics of surface markers including identifying the expression of hTERT gene in a cell, the hTERT gene-transformed neural stem cell line in vitro and in vivo proliferation and differentiation ability, and a hTERT gene-transformed neural stem cell.
  • the present invention provides a method for identifying the expression of a transduced exogenous hTERT gene in a neural stem cell line, including: (a) determining the transduction using methods such as RT-PCR, quantitative PCR, western blotting, and immunohistochemistry; The exogenous hTERT has been integrated into the chromosome of the target cell; and (b) telomerase activity and telomere length are tested, proving that hTERT has enzymatic activity and stable telomere length.
  • the present invention also provides a method for identifying the differentiation ability of hTERT-transformed neural stem cell lines, including in vitro induction and in vivo differentiation, and a method for detecting the result of differentiation.
  • the present invention provides a hTERT gene-transformed neural stem cell line for animal transplantation experiments and proves that it can be induced to differentiate into neurons, astrocytes, and oligodendrocytes in vivo.
  • the invention also provides a method for in vitro induction of hTERT gene-transformed neural stem cell lines, and differentiates into neurons, astrocytes, and oligodendrocytes.
  • the invention also provides a method for identifying the neurons, astrocytes and oligodendrocytes after the hTERT gene-transformed neural stem cell line is induced to differentiate.
  • the present invention also provides a method for identifying the hTERT gene-transformed neural stem cell line as having a proliferation capacity, including determining a cell growth curve and calculating a cell doubling time.
  • the present invention also provides a method for identifying a surface marker of a hTERT-transformed neural stem cell line.
  • the present invention provides a method for analyzing the karyotype of a hTERT transgenic neural stem cell line.
  • the invention also provides a method for measuring the virus titer.
  • the invention also provides a method for measuring the transduction efficiency of retroviruses.
  • the invention also provides a method for identifying whether a hTERT gene-transformed neural stem cell line has tumorigenicity, including, but not limited to, a method of cell soft agar clone growth experiment and a method for detecting tumorigenicity of xenogeneic animal inoculation.
  • microorganisms of the present invention have been deposited in the General Microbiology Center of the China Microbial Strain Collection Management Committee.
  • Preservation unit General Microbiology Center of China Microbial Strain Collection Management Committee; Preservation unit address: No. 13 North 1st, Zhongguancun, Ouhaidian District, Beijing, Institute of Microbiology, Chinese Academy of Sciences;
  • Figure 1 is a quantitative and quantitative RT-PCR method for quantitative comparison of Hela cells, primary cultured neurospheres, and human neural stem cell lines transfected with hTERT genes (represented in the figure as 1, 2 and 3 respectively) at the mRNA level. Case.
  • the expression level of hTERT gene at mRNA level in human neural stem cell lines transfected with hTERT gene is similar to that of Hela cells, which is about 1.5 times higher than that of primary cultured neurospheres.
  • Figure 2 shows the results obtained by detecting the telomerase activity of the human neural stem cell line transfected with hTERT using the telomere repeat amplification method (TRAP ELI SA).
  • the microplate reader measures the absorbance at 450nm and the reference at 690nm.
  • the calculated A450nm-A690nm values represent telomerase activity.
  • 1 is a 293 positive control cell
  • 2 is a primary cultured neurosphere, that is, a neural stem cell
  • 3 is a hTERT gene-transformed human neural stem cell line.
  • the bar graph shows the change in absorbance of the three cells before and after heating.
  • Figure 3 is a chromosome karyotype analysis of a human neural stem cell line showing double the normal human Somatic karyotype, 22 autosomes, one X chromosome and one Y chromosome.
  • Figure 4 shows the cloning ability of SH-SY5Y neurotumor cell lines, neural stem cells, and hTERT-transformed human neural stem cell lines in soft agar colony formation experiments, where 1 represents SH-SY5Y neurotumor cell line and 2 represents neurospheres (nerves Stem cells), 3 represents the human neural stem cell line transfected with hTERT gene.
  • the number of cloned cells of the SH-SY5Y neurotumor cell line increased a lot, and the number of hTERT-transformed human neural stem cell line monoclonal cells increased similarly to the number of neurospheres.
  • Figure 5 and Figure 6 are the results of using the RT-PCR method to identify the characteristics of the human neural stem cell line transfected with hTERT gene at the mRNA level.
  • Neural stem cell line 2 is a human neural stem cell line that is transduced with hTERT gene after induction.
  • Figure 6 is a quantitative analysis of real-time RT-PCR analysis of hTERT transgenic human neural stem cell lines before and after induction of changes in GFAP and MAP-2 mRNA levels. The histogram shows the changes in glia specific to GFAP before and after induction, and the changes before and after induction in MAP2-specific neurons. The ordinate is the logarithmic value of the mRNA copy number.
  • Figure 7 shows the results of immunohistochemical staining of hTERT-transformed neural stem cell line markers.
  • Figure 7A is GFAP (astrocyte and neural stem cell marker) immunofluorescent labeling positive cells, the cells are shown in green;
  • Figure 7B is nes tin (neural stem cell marker) positive cells, the cells are red;
  • Figure 7C is MAP2 ( Neuron mark) Weak positive cells with red cells.
  • Figures 7D-7F are the results of immunofluorescence double staining of the same neural stem cell line.
  • Fig. 7D is a GFAP-positive cell, the cell is green;
  • Fig. 7E is a nestin-positive cell, the cell is red;
  • Fig. 7F is a superposition of Fig. 7D and Fig. 7E.
  • Ruler length is 15um.
  • FIG. 8 shows the results of immunofluorescence staining of hTERT transgenic neural stem cell lines after induction with retinoic acid.
  • FIG. 8A is GFAP (astrocytic marker) positive cells induced by retinoic acid, the cells are green;
  • FIG. 8B is MAP2 (neuronal marker) positive cells induced by retinoic acid, the cells are red;
  • the lower corner of FIG. 8B is an enlarged MAP2 positive neuron.
  • FIG. 8C is 01 (oligodendrocyte marker) positive cells induced by retinoic acid, and the cells are red.
  • Figure 9 is a Western blot analysis (Wes tern b lot as say) detection of human neural stem cell lines transfected with hTERT gene before and after induction-tubu l in (54KD), Nes t in (22 OKD), GFAP (50 D), MAP2a, b (280 D) and NSE (45 D).
  • Fig. 10 shows the results of survival of 8 weeks after transplantation of a rat spinal cord injury animal model of a hTERT gene-transfected human neural stem cell line labeled with Hoechs t 33342.
  • Figs. 10A, D, and G are Hoechs t staining of a surviving neural stem cell line. The cells are all shown in blue.
  • Figure 10B is a neural stem cell line NSE (neuronal marker) positive cell, the cell is shown in red Color.
  • FIG. 10C is a double labeling of Hoechs t and NSE, and the arrows in the figure indicate double-labeled cells.
  • FIG. 10E is a neural stem cell line GFAP (Astrocyte Marker) -positive cells, and the cells are red.
  • GFAP Astrocyte Marker
  • FIG. 10H shows a 04 (oligodendrocyte-labeled) positive menstruation packet of a neural stem cell line with red cells.
  • Figure 101 shows the results of the double-labeled 04 and Hoechs t of the neural stem cell line.
  • the arrows in the figure indicate the double-labeled thin moon packs.
  • Figures 11A to 11F are immunofluorescence identifications of mouse brains after transplantation of human neural stem cell lines of the hTERT gene in adult mice.
  • the part indicated by the line segment in FIG. 11A is the injection needle path, and the other part is also the injection needle path.
  • the nucleus of the human neural stem cell transfected with hTERT gene is Ho 33342 labeled, and the nucleus is blue;
  • Figure 1 IB-labeled cells are human neural stem cell transfected with hTERT gene and differentiated into MAP2-positive neurons in vivo;
  • Figure 11C The labeled cells are mostly non-specific GFAP positive cells, and the cells are shown in green.
  • Figure 11D is a superimposed view of 11A and 11B.
  • Figure 1 1.E is an overlay of Figures 11A and 11C. No double-labeled cells were seen.
  • Figure 11F is a three-fold addition of Figures 11A, 11B, and 11C. Only Ho 33342 and MAP2 double-labeled cells demonstrate that the transplanted neural stem cell line differentiates into neurons. Detailed description of the invention
  • the present invention relates to a human neural stem cell line transfected with hTERT gene, a method for cultivating the human neural stem cell line transfected with hTERT gene, a method for identifying such a human neural stem cell line transduced with hTERT gene, and the use of the stem cell line in medicine Use in development, transplantation research, gene therapy, and various experiments.
  • This cell line can provide long-term culture of expanded human neural stem cells in vitro.
  • Primary neural stem cells can usually be extracted from embryos and adult brain tissue. Tissue sources for the isolation and purification of human neural stem cells include: neural crest, spinal cord, cerebral cortex, olfactory brain, cerebellar cortex, hippocampus, striatum, retina, and subventricular zone (SVZ). The subventricular zone SVZ is preferred in the present invention.
  • the brain tissue was separated and digested to obtain a single-cell suspension, cultured in vitro under certain conditions, and passaged to harvest neurospheres, that is, neural stem cells.
  • Culture conditions (but not limited to) 5% C0 2 , 37 ° C; DMEM / F12 (12 mg / ml) medium, basic fibroblast growth factor (10ng / ml), epidermal growth factor (20ng / ml) , Transferrin (0.1 mg / ml), sodium selenate (5.2 ng / ml), putrescine (9.6 ⁇ g / ml), insulin (0.025 mg / ml), glutamine (0 3 mg / ml), Proger terone (6.3 ng / ml), antibiotics (gentamicin 1000 U / ml).
  • Retroviruses are due to their high infection rate and stable integration. According to the procedure of purchasing a transfection kit, a retroviral recombinant plasmid carrying the hTERT gene was transfected into packaging cells, and positive clones were selected to obtain stable toxin-producing packaging cells (frozen storage). A recombinant virus carrying the hTERT gene can be obtained from the supernatant of the packaging cell culture.
  • Viral titer can be measured by inoculating tumor cell lines, including, but not limited to, Hela cells. When the tumor cells reached 60% confluence, the cells were replaced with fresh medium and cultured. The cells were digested according to 1:20, and the culture medium was replaced once every 72 hours until colonies were formed. After fixed staining, the number of colonies was counted.
  • tumor cell lines including, but not limited to, Hela cells.
  • the prepared virus supernatant can be used to infect tumor cell lines, and the resistant clones can be screened for digestion and passage, and the supernatant can be collected. After filtering, the supernatant can be used to infect non-virus-infected tumor cells again, and the resistant clones can be selected ( Cell colonies). If no cell colonies are formed, no wild-type helper virus is produced.
  • the measurement of viral transduction efficiency includes, but is not limited to, the use of a retrovirus carrying a green fluorescent protein (retroviral vector and promoter consistent with the vector carrying the gene of interest), infection of neural stem cells, and transduction subculture for 24 hours
  • a retrovirus carrying a green fluorescent protein retroviral vector and promoter consistent with the vector carrying the gene of interest
  • For neural stem cells add polybrene to the virus supernatant to a final concentration of 8 ⁇ g / ml, mix for 12 hours, and infect twice in 72 hours. After 48 hours, a drop of cell suspension smear was taken and observed under a direct fluorescence microscope. The percentage of green fluorescent positive cells in 100 cells was counted, 3 high-power fields were counted, and the average was counted 3 times.
  • hTERT recombinant retrovirus transduced neural stem cells to obtain transduced hTERT gene human neural stem cell lines, established single-cell clone lines, passaged expansions, and identified.
  • Identification of hTERT gene-transformed neural stem cell lines requires identification from several aspects, to identify the expression of transduced hTERT genes in neural stem cell lines, telomerase activity and telomere length carried by neural stem cell lines, and to identify prepared nerves. Whether the stem cell line has the ability to proliferate and differentiate and the surface markers of the neural stem cell line can be determined after the establishment of a neural stem cell line transducing the hTERT gene.
  • transduced exogenous hTERT gene was detected in hTERT transgenic neural stem cell lines.
  • the expression of transduced exogenous hTERT gene in neural stem cell lines is detected at the mRNA level, and its technologies include, but are not limited to, PCR, ReaI-time quantitative PCR technology.
  • PCR method was used to amplify the genomic DNA of neural stem cell lines and observe the presence of specific bands to determine the expression of hTERT gene in the genome of neural stem cell lines.
  • Rea l-ime quantitative PCR technology was used to quantitatively compare hTERT gene mRNA expression in tumor cells, neurospheres (neural stem cells) and hTERT gene-transfected neural stem cell lines.
  • telomerase activity in hTERT-transfected neural stem cell lines Not limited to, PCR ELISA technology.
  • the cell extract was prepared and subjected to a PCR reaction for 30 cycles, followed by probe hybridization, ELISA color development, and a microplate reader to calculate the A450nm-A690nm value table.
  • the telomerase activity is shown. The difference between the two represents the telomerase activity.
  • the method for determining the telomere length of hTERT-transfected neural stem cell lines uses, but is not limited to, the TRF Southern hybridization technique.
  • HTERT gene-transformed neural stem cell lines can be tested for differentiation, including induction of differentiation in vitro and in vivo, and detection of differentiation results.
  • HTERT transgenic neural stem cell lines were induced in vitro.
  • Cytokines include IL-1, IL-11, GDNF, LIF, bFGF2, PDGF, CNTF, T3, BDNF, etc.
  • the chemical reagent was retinoic acid.
  • Neural stem cell lines were transplanted in animal models to detect their differentiation in vivo. Neural stem cells can be differentiated into neurons, astrocytes, and oligodendrocytes. The hTERT-transformed neural stem cell line can be differentiated into the above three types of cells, which is a marker for detecting the differentiation ability of the neural stem cell line.
  • Neural stem cell markers detected include nes tin in, GFAP; neuron markers include ⁇ -tubul 1 in III, MAP2, MAP 5, neurofilament proteins, NeuN, NSE; astrocytes are labeled with GFAP; oligodendrocytes Plasma cells include 01 and 04. Detection methods include real-time RT-PCT, immunofluorescence staining and Western blotting.
  • Methods for identifying the proliferation capacity of hTERT-transformed neural stem cell lines include cell growth curve determination and calculation of cell doubling time.
  • the growth curve of neural stem cells is an important indicator for detecting the proliferation of neural stem cells during the survival period of a generation. It is a coordinate graph with culture time as the abscissa and cell density as the ordinate.
  • the calculation method of hTERT gene-transformed neural stem cell line population doubling time can only be used to estimate the cell population doubling time. It takes time for the cells to adapt to the new culture environment after inoculation, which is called the stationary phase. After 24 hours it began to divide and proliferate again. Calculating the cell population doubling time is best to let the neural stem cell line grow in a plastic petri dish for 7 days, that is, after the seventh day of growth, the cells can cover 90% -95% of the lone culture area.
  • Method for identifying surface markers of hTERT transgenic neural stem cell lines Adherent neural stem cells are digested to prepare a single cell suspension. Cell surface markers were detected by flow cytometry. Detection surface antigens include CD133, CD90, CD71, CD117, CD34, and CD45. For the karyotype analysis of neural stem cell lines, the karyotype G-band analysis method was used. Methods for identifying whether a neural stem cell line is tumorigenic include, but are not limited to, a cell soft agar clone growth test and a method for detecting tumorigenicity of xenogeneic animals.
  • the invention provides a hTERT gene-transformed neural stem cell line, a method for establishing the same, and a related identification method.
  • the hTERT gene-transformed neural stem cell lines provided by the present invention not only can adapt to in vitro growth, but also can be expanded in large quantities in vitro, and have no phenotypic changes or carcinogenicity. They have adherent growth, very similar to primary cells, Contact inhibition, serum and growth factor-dependent traits, no chromosomal changes, and the ability to differentiate neural stem cells, can differentiate into neurons, astrocytes, and less in animal experiments and in vitro induction experiments Dendritic cells. Therefore, this hTERT transgenic neural stem cell line is of great significance for medical research.
  • HTERT transgenic neural stem cell lines can be used as a cell platform for neural stem cell research in basic research. It has clinical potential for in vivo transplantation research and treatment of patients.
  • the neural stem cell line can be transplanted into the brain or spinal cord of an animal such as a rat, a mouse or a monkey, and can be used for the study of nervous system tumors, degenerative changes and injuries.
  • the neural stem cells have the ability to replace dead neurons in neurodegenerative lesions.
  • the neural stem cell line carries a certain trophic factor gene, a cytokine gene, or a special gene for gene therapy.
  • a method of treating a patient includes administering the neural stem cell line to the patient.
  • the subventricular zone (SVZ) was separated, digested with 0.01% EDTA and 0.25% trypsin for 3 minutes, and cultured with DMEM / F12 containing 10% fetal bovine serum Digestion was terminated. After centrifugation, single-cell suspension culture was performed by mechanical pipetting, and medium was added, and cultured at 5% CO 2 and 37 ° C.
  • neural stem cell spheres were selected, that is, neural stem cells.
  • the subventricular zone (SVZ) forms nerve cell spheres in 2-3 days.
  • the media conditions are:
  • bFGF basic fibroblast growth factor
  • EGF epidermal growth factor
  • Antibiotic Gentamicin 1000 U / ml
  • the digested plasmid DNA was subjected to 0.8% agarose electrophoresis. After the hTERT fragment and the carrier fragment were completely separated, the hTERT cDNA agarose gel band was harvested under a 360 nm UV lamp, and placed in a 1.5 ml centrifuge tube with a sealing membrane.
  • Packaging cells PT67 were cultured in DMEM medium containing 10% fetal bovine serum at 5 ° /. C02, 37 ° C constant temperature culture. Digest and pass once every 1-2 days.
  • Cell preparation Inoculate 5 x 105 5 PT67 cells in 50ml culture flasks 24 hours before transfection, and culture at 37 ° C to 60% confluence. Change the fresh medium and incubate for 2-3 hours for transfection.
  • Fugene 6 wraps plasmid DM Take two autoclaved Ep tubes, add 2 ⁇ g of DNA and 6 ⁇ 1 Fugene 6, and add serum-free DMEM 194 ⁇ 1 to the second tube. Leave it at room temperature for 5 minutes, slowly add the mixture of transfection reagent and DMEM to the DNA tube, and mix thoroughly. Leave at room temperature for 15 minutes. 3. Transfection: Add the above transfection reagent and DNA mixture to the cell culture medium and incubate without removing the serum from the culture medium and changing the culture medium. Incubate at 37 ° C and 5% CO2, and gently shake the culture bottle every 30 minutes until After 48 hours the cells were passaged 1:10 and replaced with Puromyc in selection medium.
  • Amplification of positive clones Select positive clones, digest them with trypsin, and transfer them to different culture flasks. Puromyc in selection medium is maintained for 7-10 days and then removed. Supernatants are collected after the flask is full, 0.45 ⁇ After filtration, the filter was stored at -70 ° C for future use, and the frozen packaging cells were used for future use. At this time, a recombinant retrovirus carrying the hTERT gene was obtained.
  • Hela cells were used to detect the virus titer in the above-prepared packaging cell supernatant. The higher the titer, the more powerful the virus is.
  • Inoculate 5 X 10 4 Hela cells in a 6-well plate change the fresh medium when it reaches 60% confluence, and incubate for 2 hours, add 2ml of virus supernatant containing 10 2 , 10 3 , and 10 4 dilutions, and add po lybrene Cultivate at a concentration of 8 ug / ml at 37 ° C, gently shaking the flask every 30 minutes.
  • the culture solution was added to dilute the polybrene concentration to 2 ⁇ g / ml.
  • the culture was continued for 48 hours, and the passaged cells were digested at 1:20, and Puromyc in was added to 3 ug / ml.
  • the supernatant was discarded, fixed in cold methanol, and stained with Giemsa, and the number of colonies was counted under a microscope. Each well was replicated in 3 wells and averaged.
  • the titer calculation method is as follows:
  • Hela cells were infected with the retroviral supernatant prepared in Example 3. Puromyc in screened resistant clones were digested and passaged. Supernatants were collected after the bottle was full. After filtering through a 0.45um filter, the supernatant was re-infected without infection. Hela cells of the virus were screened for resistant clones by Puromyc in, and no cell colonies were formed, indicating that no wild-type helper virus was produced. Example 6. Measurement of retroviral transduction efficiency
  • Example 7 hTERT recombinant retrovirus transduces neural stem cells to obtain hTERT gene-transformed neural stem cell lines.
  • the transduction efficiency was determined in the same manner as in Example 6. After repeated infection, the cells were cultured for another 72 hours. ml of puromycin was screened, fresh selection medium was changed every three days, and primary cultured neural stem cells without any virus were used as a control, and maintained for 14 days. Growth of drug-resistant cell clones can be observed within 1-2 weeks. After 2-3 weeks, clones with good isolation can be selected by cloning rings. A hTERT-transformed neural stem cell line was obtained.
  • Example 9 The genomic DNA of the hTERT gene-transformed neural stem cell line prepared in Example 7 was extracted for PCR amplification, and the cell genome D hTERT amplified a specific band, indicating that the hTERT cDNA was integrated in the neural stem cell genome.
  • Example 9 Real-time quantitative PCR was used to compare the expression of hTERT gene at mRNA level in Hela cells, neural stem cells, and neural stem cell lines transfected with hTERT genes.
  • cMA template Use 1 ⁇ 1 of cMA template, add 0.5 ⁇ 1 (10pmol) of upstream and downstream primers; 0.3 ⁇ 1 (10 ⁇ 1) of TaqMan fluorescent probe; 0.15 ⁇ 1 (5 units) of Gold Taq DNA polymerase; 1 ⁇ 1 of TaqMan buffer; 25mM MgCl 2 1.2 ⁇ 1; Add double distilled water to 15 ⁇ 1. Perform PCR reaction on ⁇ 7700 quantitative PCR instrument.
  • GAPDH standard curve preparation and data processing 500 ng, 50 ng, 100 ng, 50 ng, 25 ng, 10 ng, 5 ng, 1 ng, 500 pg, 50 pg, and 5 pg total RNA were reverse transcribed to make cDNA, and then each ⁇ ⁇ ⁇ cDNA Do Real-Time PCR. Four concentrations of Ct were measured at each concentration and the arithmetic mean and standard deviation were taken. A standard curve was obtained by linear regression using Oginal software. The measured C t value of the sample cDNA is compared with this standard curve to calculate the sample The amount of mRNA relative to the template.
  • telomerase PCRELISA PCRELISA to detect telomerase activity of hTERT gene-transfected neural stem cell lines
  • PCR reaction Take 25ul reaction mixture, add 1-3 ul cell extraction solution, and add sterile water to a final volume of 50 ul. Reaction conditions: 25 ° CX30 minutes; 94 ° CX5 minutes; 94 ° CX30 seconds; 50 ° CX30 seconds; 72 ⁇ 90 seconds, 30 cycles, 72 ° CX10 minutes.
  • Probe hybridization and ELISA color development Mix 20ul Denaturation reagent and
  • the microplate reader measures the absorbance at 450nm and the reference at 690nm.
  • the calculated A450nm-A690nm values represent telomerase activity.
  • Example 11 The telomere length of a hTERT-transfected neural stem cell line was detected by a TRF Southern hybridization method. The results showed that the neural stem cell line had a stable telomere length.
  • Example 13 Determination of growth curve of hTERT-transformed neural stem cell lines
  • Culture cells The same number of hTERT gene-transfected neural stem cells (21 bottles if culture flasks are used) are seeded into 21 well plates. Count and record the density of seeded cells (1 ⁇ 10 4 cells / ml according to the rate of cell proliferation). It is best to use a 6 cm diameter plastic petri dish and add 5 ml of culture solution. The inoculation time was recorded as 0 hours.
  • Count cell density Count the cell density in 3 wells every 24 hours from the time of seeding, count the cells by trypsin digestion, and calculate the average and standard deviation of the number of cells. To improve accuracy, cells can be counted 2-3 times per well. This was done until the end of the seventh day.
  • the number of cells initially seeded is 5 cells per lxio.
  • the cell proliferation can cover 90%-95% of the petri dish area, trypsinize and count the number of cells.
  • hypotonic treatment of cells Dissolve the cells in 5ml of hypotonic solution (0.075M KC1), and place them in a 37 ° C incubator for 30 minutes.
  • Dropper Pipette the appropriate cell suspension with a pipette and drop it over a long distance onto a glass slide washed with distilled water.
  • Figure 3 shows that the neural stem cells obtained have a normal human diploid karyotype, which contains 22 pairs of autosomes, one X chromosome, and one Y chromosome.
  • Clone count Cell clone count can be performed after 2-3 weeks of culture. A cell cluster consisting of more than 40 cells is one clone. The number of cells within the clones increased as the culture time increased. If the cells are fine-grained, the outline of the cells is unclear, indicating that the cells have died. Observation with a microscope showed that the cells in the clones were round, translucent, and arranged into clusters.
  • the results are shown in Figure 4.
  • the SH-SY5Y neural tumor cell line has a strong ability to form clones, which is a hallmark of tumor cells.
  • the cloning ability of hTERT-transformed neural stem cell lines is similar to neural stem cells. Not tumorous.
  • Example 17 Detection of tumorigenicity of heterogeneous animals inoculation
  • Neural stem cell lines were digested and plated into 6-well plates with 1 ⁇ 107 dishes (some dishes were covered with coverslips). After the cells adhered to the wall, the inducer all-trans retinoic acid (2uM) was added for 18 days.
  • Example 19 Identification of mRNA-level characteristics of hTERT-transformed neural stem cell lines by RT-PCR
  • Reverse transcription was performed according to a system of 3-5 ⁇ ⁇ / 20 ⁇ 1.
  • the first-strand cDNA was synthesized by the M-MLV method. Then add buffer and RNase inhibitor, mix well, Incubate at 37 ° C for 2 minutes, then add the reverse transcriptase M- MLV, incubate at the appropriate temperature, and finally terminate the reaction.
  • the prepared cDNA can be used in the following PCR reaction.
  • the template cDNA obtained in the above experiment 2.5 mM dNTP 2 ⁇ 1, upstream and downstream primers 1 ⁇ (lOpmol), Taq enzyme 1 ⁇ 1, lOxBuf fer (20mMMgCl 2 ) 2.5 ⁇ 1, ddH 2 0.
  • the reaction was: 95 ° C 10 minutes, ( 30 seconds at 95 ° C, 45 seconds at 57 ° C, 1 minute at 72 ° C) 40 cycles, 10 minutes at 72 ° C, 4 ° C.
  • RT-PCR quantitative analysis of RT-PCR was performed first.
  • Column 1 is the hTERT gene-transformed neural stem cell line before induction
  • column 2 is the hTERT gene-transformed neural stem cell line after induction.
  • Further real-time RT-PCR quantitative analysis was performed.
  • Glial cells specifically labeled with GFAP decreased after induction
  • neuronal cells specifically labeled with MAP2 increased after induction.
  • this neural stem cell line Can differentiate into neurons, astrocytes and oligodendrocytes.
  • Example 20 Method for detecting markers of neural stem cell line by immunofluorescence
  • Primary antibody reaction Add the primary antibody of appropriate dilution and incubate for 1 hour at room temperature in a wet box.
  • Secondary antibody reaction Add secondary antibody (FITC or rhodamine labeled) at a dilution of 1: 50, and incubate in the chamber at room temperature for 1 hour.
  • secondary antibody FITC or rhodamine labeled
  • Fluorescence test solution 8.5ml glycerol + 500ul p-phenylenediamine (20mgAml), adjust the pH to 8.0 with carbonate buffer.
  • Example 21 Wes tern blot determination of protein expression differences in neural stem cell lines before and after retinoic acid induction
  • Blocking Block the solution with 5% skimmed milk powder and block for 1 hour at room temperature on a shaker (blocking solution is prepared with TBST).
  • Secondary antibody binding Dilute the secondary antibody working solution (dilute the secondary antibody with blocking solution at 1: 1000, The secondary antibody used in the chemiluminescence method was linked with horseradish peroxidase) and a nitrocellulose membrane in a hybridization bag, and incubated at room temperature for 1 hour on a shaker.
  • Color development Use the chemiluminescence color development reagent of NEN LIFE SCIENCE for color development. Take equal volumes of oxidant and luminous enhancer and mix in a plate. The hybridization surface of the nitrocellulose membrane was brought into full contact with the color development solution, and the color development results were detected using a Kodak 440CF imaging system.
  • FIG. 9 Western blot chemiluminescence was used to detect the expression of the following proteins before and after retinoic acid-induced neural stem cell lines transfected with hTERT: a- tubulin (54KD), Nestin (220 D), GFAP (50 KD), MAP2a , b (280KD) and NSE (45 KD).
  • 1 represents a hTERT gene-transformed neural stem cell line
  • 2 represents a hTERT gene-transformed neural stem cell line induced by retinoic acid.
  • neural stem cells were identified at the protein level.
  • Nestin and GFAP were expressed before induction, and GFAP and MAP2 were expressed after induction, which proved that the cells we obtained have neural stem cell characteristics.
  • Nestin is a marker of neural stem cells
  • MAP2 is a marker of neurons
  • GFAP is a marker of astrocytes.
  • hTERT-transformed neural stem cell lines can differentiate into neurons, astrocytes, and oligodendrocytes in the rat spinal cord.
  • microorganism (strain) hexyl 2002 1 () 22 January received by the Collection, and were registered. According to your request, it will be kept for 30 years from that date, and it will be kept for another 5 years after receiving the request to provide microbial samples before the expiration.

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Abstract

La présente invention concerne les cellules souches neuronales humaines transduites par le gène hTERT, ainsi que la méthode de production et d'identification de ces dernières. La méthode comprend les étapes suivantes: (1) l'extraction de cellules souches neuronales humaines, l'incubation avec expansion in vitro de manière à obtenir des sphères neuronales, c'est-à-dire des cellules souches neuronales; (2) le conditionnement du virus de recombinaison porteur du gène hTERT; (3) la transfection des cellules souches neuronales avec le virus de recombinaison porteur du gène hTERT; et (4) le repiquage et l'expansion des cellules souches neuronales humaines transduites par le gène hTERT de manière à obtenir des lignées de cellules souches neuronales humaines. L'activité télomérase des cellules souches neuronales et la longueur des télomères sont identifiées ainsi que les propriétés des cellules souches neuronales. La production de lignées de cellules souches neuronales est établie. Les cellules souches neuronales peuvent être utilisées pour l'étude fondamentale et pour le criblage de médicaments destinés au système neuronal et possèdent une grande valeur clinique, on peut également les utiliser en tant que vecteur cellulaire dans la thérapie génique des maladies du système neuronal, pour les maladies neurodégénératives des systèmes neuronaux et en tant que matériaux cellulaires de transplantation pour la réparation des lésions.
PCT/CN2002/000745 2002-10-23 2002-10-23 Cellules souches neuronales humaines transduites par le gene htert, methode de production et d'identification de ces dernieres WO2004038011A1 (fr)

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PCT/CN2002/000745 WO2004038011A1 (fr) 2002-10-23 2002-10-23 Cellules souches neuronales humaines transduites par le gene htert, methode de production et d'identification de ces dernieres

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999050407A1 (fr) * 1998-03-26 1999-10-07 Kyowa Hakko Kogyo Co., Ltd. Anticorps monoclonal contre une sous-unite catalytique de telomerase humaine
WO2003001198A1 (fr) * 2001-06-21 2003-01-03 Regents Of The University Of California Immortalisation de cellules humaines par expression ectopique de telomerase transcriptase inverse
WO2003014320A2 (fr) * 2001-08-10 2003-02-20 Cornell Research Foundation, Inc. Cellules souches neuronales humaines immortalisees issues de la telomerase et cellules souches a restriction phenotypique

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999050407A1 (fr) * 1998-03-26 1999-10-07 Kyowa Hakko Kogyo Co., Ltd. Anticorps monoclonal contre une sous-unite catalytique de telomerase humaine
WO2003001198A1 (fr) * 2001-06-21 2003-01-03 Regents Of The University Of California Immortalisation de cellules humaines par expression ectopique de telomerase transcriptase inverse
WO2003014320A2 (fr) * 2001-08-10 2003-02-20 Cornell Research Foundation, Inc. Cellules souches neuronales humaines immortalisees issues de la telomerase et cellules souches a restriction phenotypique

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