WO2018113145A1 - 一种同时抵抗细胞衰老及恶性转化的人多能干细胞的制备方法 - Google Patents

一种同时抵抗细胞衰老及恶性转化的人多能干细胞的制备方法 Download PDF

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WO2018113145A1
WO2018113145A1 PCT/CN2017/080393 CN2017080393W WO2018113145A1 WO 2018113145 A1 WO2018113145 A1 WO 2018113145A1 CN 2017080393 W CN2017080393 W CN 2017080393W WO 2018113145 A1 WO2018113145 A1 WO 2018113145A1
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nrf2
enhanced
cell
stem cells
gene
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刘光慧
曲静
杨济平
铃木敬一郎
任若通
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中国科学院生物物理研究所
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    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
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Definitions

  • the invention belongs to the field of biotechnology and relates to a preparation method of human pluripotent stem cells which simultaneously resist cell senescence and malignant transformation.
  • Cell transplantation therapy is the most promising and potential development direction in the field of regenerative medicine in recent years.
  • Cell transplantation is intended to supplement or replace cells that have been depleted or lose normal function due to factors such as disease or aging.
  • the transplanted cells survive and function in the body, restore tissue homeostasis, and thereby achieve tissue repair and regeneration.
  • cell transplantation therapy has shown excellent therapeutic effects in the treatment of leukemia such as bone marrow stem cell transplantation, its wider application still faces many challenges. At present, the main factors limiting cell transplantation therapy focus on both effectiveness and safety.
  • the effectiveness of cell transplantation therapy is mainly reflected in the quality of the graft before transplantation and whether it can survive and integrate and regenerate after entering the body.
  • the graft has a certain "life" in the in vitro culture process, which is characterized by continuous exposure to replication stress, and after a limited continuous passage, it rapidly enters the cellular senescence state, stops proliferation and loses function.
  • the site of cell transplantation is generally located in the tissue microenvironment of disease or aging, in which inflammatory factors, harmful metabolites, etc. damage the exogenous graft. Both of these aspects can lead to severe functional decline in cells, resulting in inefficient transplantation.
  • the safety of cell transplantation therapy is mainly focused on the tumorigenic risk after long-term transplantation.
  • Cell grafts continue to be stressed by stress in the body, and the genome may become extremely unstable. If a mutation occurs in an oncogene or a tumor suppressor gene, it may become a cancer, posing a safety hazard.
  • gene-targeted editing technology has greatly promoted the progress of cell transplantation therapy. This technology allows people to accurately edit the genetic code on the genome. A number of cases have been reported to correct disease-causing mutations in disease cells using gene-targeted editing techniques. Gene correction cell External amplification can be used for autologous transplantation and is an excellent material for cell transplantation therapy.
  • the method for preparing mesenchymal stem cells comprises the following steps:
  • the method of mutating the glutamic acid at position 82 of the NRF2 protein in the human pluripotent stem cell to glycine is to mutate the base A of the 245th position of the NRF2 gene. Is the base G; the 245th position of the NRF2 gene is position 245 of the NRF2 gene coding region.
  • the method of mutating the base A of the 245th position of the NRF2 gene into the base G is genome-based point editing.
  • the method of genomic fixed point editing may be ZFN editing, TALEN editing, CRISPR/Cas9 editing or HdADV mediated fixed point editing.
  • the method of genomic fixed-point editing is HdADV-mediated fixed-point editing.
  • the HdADV mediated fixed point editing can be implemented by:
  • the first step the acquisition of mutant fragments
  • nucleotide position of position 245 corresponding to the cDNA sequence of the NRF2 gene in the gene fragment of the human genome NRF2 gene intron 1 and intron 2 was mutated from the wild type base A to a base G, and keeping the other sequences unchanged, to obtain a fragment after the mutation;
  • the second step the construction of the virus expression plasmid
  • the mutated fragment was inserted into the AscI and SpeI cleavage sites of the pCIHDAdGT8-4 vector to obtain a recombinant plasmid.
  • the virus expression plasmid and the helper plasmid are co-transfected into packaging cells to obtain recombinant adenovirus particles.
  • the recombinant plasmid was digested with PI-SceI (NEB) to obtain a linearized plasmid, and the linearized plasmid was introduced into a packaging cell together with the helper virus AdHPBGF35 to carry out recombinant adenovirus packaging to obtain recombinant adenovirus particles;
  • the cells are specifically derived cell line 116 cells of human embryonic kidney cell line 293;
  • the fourth step the recombinant adenovirus particles infect the target cells
  • NRF2 AG/+ hESC cells Human pluripotent stem cells were infected with recombinant adenovirus particles, and a cell with the correct editing of the NRF2 allele was screened and designated as NRF2 AG/+ hESC cells;
  • the linearized plasmid and the helper virus AdHPBGF35 are co-introduced into the NRF2 AG/+ hESC cells for gene editing, and the edited cells are identified to obtain correct positions on the two NRF2 alleles.
  • the edited cell is the NRF2 gene-enhanced pluripotent stem cell.
  • the method for directionally inducing differentiation comprises the following steps:
  • the specific method of the embryoid body differentiation is as follows: a NRF2 AG/AG hESC clone containing 300-500 cells and uniform size is prepared, washed once with room temperature PBS, and then digested with Dispase at 37 ° C for 20-30 min. After the ESC clones were formed into spheres, they were resuspended in CDF12 medium, and then added to a low adhesion culture plate (Corning, Inc., Cat. No. 3471), and embryoid bodies were formed after 1-3 days of incubation at 37 ° C under 5% CO 2 conditions.
  • the specific method for culturing the embryoid body to the appearance of fibrilla cells is as follows: the embryoid body is inoculated into a Matrigel-coated 6-well plate for culture, and culture is continued for 2 weeks until the appearance of fibrillar cells.
  • the human pluripotent stem cell is a human embryonic stem cell or a human induced pluripotent stem cell.
  • the human embryonic stem cell is specifically a human embryonic stem cell H9 cell line.
  • Another object of the present invention is to provide a NRF2 gene-enhanced mesenchymal stem cell obtained by the above method.
  • Still another object of the present invention is to provide a novel use of the above-described NRF2 gene-enhanced mesenchymal stem cells or the above-described NRF2 gene-enhanced pluripotent stem cells.
  • the present invention provides the use of the above NRF2 gene-enhanced mesenchymal stem cells or the above-described NRF2 gene-enhanced pluripotent stem cells in any of the following c1)-c5):
  • the present invention also provides the use of the above NRF2 gene-enhanced mesenchymal stem cells or the above NRF2 gene-enhanced pluripotent stem cells in any of the following d1)-d5):
  • a final object of the invention is to provide a product.
  • the active ingredient of the product provided by the present invention is the above-mentioned NRF2 gene-enhanced mesenchymal stem cells
  • M1 resist and/or delay cell senescence
  • n1)-n6 The resistance and/or retardation of cellular senescence is manifested in any of the following n1)-n6):
  • the gene associated with cell senescence is specifically a p16 gene, a p21 gene, and a GATA4 gene;
  • N5 has a more complete nuclear structure
  • the cell transplantation treatment is a cell transplantation repair treatment.
  • Figure 1 shows a pluripotent stem cell producing a single site editing of the resistance gene NRF2, and thereby gene-enhanced mesenchymal stem cells, in which NRF2 activity is continuously activated.
  • A is the pattern diagram of gene editing;
  • B is the sequencing result of A245G locus of NRF2 gene;
  • C is the morphology of obtained pluripotent stem cells and immunofluorescence identification of trigeminal differentiation in vivo;
  • D is the dry gene of pluripotent stem cells Expression of E; is the identification of surface markers associated with MSC in differentiated MSCs;
  • F is the content of NRF2 protein in nucleus and nucleus in MSC;
  • G is the mRNA level of NRF2 downstream target gene;
  • H is NRF2 and Protein expression of key downstream target genes NQO1 and HO-1.
  • Figure 2 shows the characteristics of gene-enhanced mesenchymal stem cells against stress and cellular senescence.
  • A is the detection of cell viability under the treatment of exogenous irritants;
  • B is the growth curve of MSC;
  • C is the result of SA- ⁇ -Gal staining;
  • D is the detection of reactive oxygen species;
  • E is the result of the expression of senescence-related markers; Detection of cell proliferation-associated marker Ki67;
  • G is nuclear membrane integrity assay.
  • Figure 3 shows the characteristics of gene-enhanced mesenchymal stem cells against malignant transformation.
  • A is a pattern of malignant transformation in vitro;
  • B is the non-anchor-dependent growth of MSC after malignant transformation;
  • C is the tumorigenic ability of MSC after malignant transformation;
  • D is the longitudinal and longitudinal section of mouse long tumor section.
  • Figure 4 is an example of transplantation of gene-enhanced mesenchymal stem cells for blood flow recovery in a hind limb ischemia model.
  • A is the retention ability of MSC in the muscle microenvironment
  • B is the blood flow recovery of each hind limb ischemic mouse
  • C is the representative picture of the mouse leg at different time points.
  • CDF12 medium formula (1) CDF12 medium formula:
  • DMEM/F12 medium (Invitrogen, 11320-033);
  • MSC Mesenchymal stem cell
  • JPC human fibroblast growth factor
  • the MSC differentiation medium requires an additional 5 ng/ml TGF ⁇ (Humanzyme, HZ1131).
  • the cell line is as follows:
  • the human embryonic stem cell H9 cell line is a product of WiCell, Inc., number: WA09(H9)-DL-7.
  • Human embryonic kidney cell 293T was purchased from ATCC under the accession number CRL-3216.
  • the culture method of human embryonic stem cell H9 cell line is as follows:
  • H9 cells were seeded in a culture plate previously coated with an extracellular matrix (qualified-Matrigel, BD Biosciences, USA, Cat. No. 354277), and cultured using mTeSR medium (product of StemCell Technologies, USA).
  • the biological materials used for virus packaging are as follows:
  • the biological materials used in the packaging of recombinant adenovirus are as follows:
  • the pCR2.1-TOPO vector is a product of Invitrogen.
  • helper adenoviral vector pCIHDAdGT8-4 vector see "An HSV amplicon-based helper system for helper-dependent adenoviral vectors. Shuji Kubo, et al. BBRC. 2003. 307 (4): 826-830", the public can be from the original author Or obtained from the Institute of Biophysics of the Chinese Academy of Sciences.
  • Transfected human embryonic kidney cell line 293 derived cell line 116 cells see "Improved system” For helper-dependent adenoviral vector production. Palmer D. and Ng P. Molecular Therapy. 2003.8 (5): 846-52.” The article is available to the public from the original author or the Institute of Biophysics of the Chinese Academy of Sciences.
  • Adenovirus AdHPBGF35 See "Genome Size and Structure Determine Efficiency of Postinternalization Steps and Gene Transfer of Capsid-Modified Adenovirus Vectors in a Cell-Type-Specific Manner. Dmitry M. Shayakhmetov, et al., Journal of Virology. 2004.78" 18): 10009-10022.” The article can be obtained from the original author or the Institute of Biophysics of the Chinese Academy of Sciences.
  • both the retroviral vector and the packaging plasmid were purchased from Addgene under the following numbers: pBABE-neo-hTERT (1774), pBABE-zeo-large T genomic (1778), pBABE-puro-HRAS V12 (1768), gag/pol (14887). ), VSV-G (8454).
  • Lentiviral vector plasmid overexpressing luciferase luciferase see "SIRT6safeguards human mesenchymal stem cells from oxidative stress by coactivating NRF2. Pan et. al,. Cell research. (2016) 26: 190-205.” Obtained from the Institute of Biophysics, Chinese Academy of Sciences.
  • P53 knockdown of the lentiviral vector plasmid PLVTHM-shP53 see "PTEN deficiency reprogrammes human neural stem cells towards a glioblastoma stem cell-like phenotype. Duan et. al,. Nature communication. (2015) 10.1038.” Obtained from the Institute of Biophysics, Chinese Academy of Sciences.
  • the lentiviral packaging plasmid was purchased from Addgene under the following numbers: psPAX (12260), pMD2.G (12259).
  • Fluorescein FITC-labeled anti-human cell surface recognition molecule CD90 antibody BD Biosciences, Cat. No. 555595.
  • Fluorescein PE-labeled anti-human cell surface recognition molecule CD73 antibody BD Biosciences, Cat. No. 550257.
  • Fluorescein APC-labeled anti-human cell surface recognition molecule CD105 antibody BD Biosciences, Cat. No. 17-1057-42.
  • Fluorescein APC labeled isotype control antibody BD Biosciences, Cat. No. 555751.
  • Fluorescein PE labeled isotype control antibody BD Biosciences, Cat. No. 555749.
  • Fluorescein FITC labeled isotype control antibody BD Biosciences, Cat. No. 555742.
  • the antibodies used for immunofluorescence are as follows:
  • Anti-human SMA antibody Zhongshan Jinqiao, article number: ZM-0003.
  • Anti-human LAP2 antibody BD Biosciences, Cat. No.: 611000.
  • Anti-Human Ki 67 antibody Vector Laboratories, Cat. No.: VP-RM04.
  • Anti-human P16 antibody BD Biosciences, Cat. No.: 550834.
  • Anti-human P21 antibody Cell Signaling Technology, catalog number: 2947.
  • the present invention makes the 245th position of the cDNA sequence of the NRF2 gene (GeneBank ID: 4780) from the wild type by targeted editing of a single site of the human stress resistance gene NRF2 in the pluripotent stem cell (PSC).
  • the base A mutation is the base G, and the 82nd position of the amino acid sequence of the NRF2 protein is changed from glutamic acid (E) to glycine (G) to obtain NRF2 enhanced pluripotent stem cells.
  • the specific steps are as follows (see Figure 1A for specific steps):
  • a gene fragment spanning the intron 1-exon2-intron2 of the human genome NRF2 gene was first obtained by PCR amplification from the RP11-483K11BAC DNA library (BACPAC Resources). Then, the nucleotide position (A245G) at position 245 of the cDNA sequence corresponding to the NRF2 gene on the fragment was mutated by a site-directed mutagenesis kit (Invitrogen, Cat. No. A13282), and the base A of the wild type was mutated to the base G.
  • the virus expression plasmid and the helper plasmid are co-transfected into packaging cells to obtain recombinant adenovirus particles.
  • the recombinant plasmid obtained in the first step was digested with PI-SceI (NEB) to obtain a linearized plasmid, and the linearized plasmid was introduced into the human embryonic kidney cell line 293-derived cell line 116 cells together with the helper virus AdHPBGF35 for recombinant adenovirus.
  • the recombinant adenoviral particles were obtained by packaging and purification by ultracentrifugation.
  • hESCs cells H9 were infected with recombinant adenovirus particles (1 ⁇ 10 7 cells, and the amount of virus used was 15bgal-transducing units (btu).
  • btu 15bgal-transducing units
  • the literature “Palmer, DJ&Ng, P.Physical and infectious titers of helper- Dependent adenoviral vectors: a method of direct comparison to the adenovirus reference material. Mol Ther 10,792-798 (2004).” and "Suzuki, K. et al.
  • PCR primers were NRF2A245GseqF: ACCATTTGTGACTTTGCCCTTTAGTGACCTCTACCATC and NRF2A245GseqR: AACCTGCCATAACTTTCCCAAGAACTGA, sequencing primer was aaaaacagaaaaaacttgaa), and a NRF2 was obtained. Cells that are correctly edited on the locus, This was recorded as NRF2 AG/+ hESC cells.
  • NRF2 AG/AG hESC cells ie NRF2 enhanced pluripotent stem cells.
  • NRF2 enhanced pluripotent stem cells The sequencing confirmed that the A245G site of the NRF2 gene in NRF2 enhanced pluripotent stem cells was correctly edited (Fig. 1B). The 245th position of the two homologous chromosomes of the NRF2 gene in NRF2 enhanced adult cells was from base A. Mutated to base G.
  • NRF2 enhanced pluripotent stem cells Morphological identification of NRF2 enhanced pluripotent stem cells, and the morphological identification results of NRF2 AG/AG hESC are shown in Fig. 1C. In addition, it can be seen from the figure that NRF2 enhanced pluripotent cells still have the ability to differentiate into inner and outer trigeminal cells in vivo.
  • NRF2 +/+ , NRF2 AG/+ hESC and NRF2 AG/AG hESC cells normally express pluripotent genes OCT4, SOX2 and NANOG.
  • the present invention further differentiates the NRF2-enhanced pluripotent stem cells obtained in Example 1 into NRF2-enhanced mesenchymal stem cells (NRF2-enhanced MSCs). It has been proved by experiments that NRF2 in NRF2 enhanced MSCs is actively activated, showing anti-reverse, anti-cell senescence and resistance to malignant transformation in vitro and in vivo. Specific steps are as follows:
  • NRF2 AG/AG hESC for embryoid body (EB) differentiation the specific steps are as follows: Prepare NRF2 AG/AG hESC clone containing 300-500 cells and uniform size, wash once with room temperature PBS, use Dispase (Invitrogen , Item No. 17105041) Digested at 37 ° C for 20-30 min. After the ESC clone was formed into a sphere, it was resuspended in CDF12 medium, added to a low adhesion culture plate (Corning, Cat. No. 3471), and cultured at 37 ° C, 5% CO 2 for 1-3 days to form a embryoid body.
  • a low adhesion culture plate Corning, Cat. No. 3471
  • the embryoid bodies obtained in the step 1 were inoculated in a matrigel (Invitrogen)-coated 6-well plate for culture, and the culture was continued for 2 weeks until the appearance of fibrillar cells. After one passage, the cell group in which CD73, CD90 and CD105 were positive was sorted by flow cytometry, which is NRF2 enhanced MSC, which was designated as NRF2 AG/AG MSC.
  • the NRF2 AG/AG hESC in the above step was replaced with the wild-type human embryonic stem cell H9 cell line, and the other steps were unchanged.
  • the wild-type mesenchymal cells were induced to differentiate into wild-type mesenchymal cells and recorded as NRF2 +/+ MSC.
  • the surface markers of NRF2 AG/AG MSC and NRF2 +/+ MSC obtained in the first step were detected.
  • NRF2 AG/AG MSC and NRF2 +/+ MSC obtained in step one can express MSC-specific surface markers CD73, CD90 and CD105, but not unrelated CD34, CD43 and CD45.
  • NRF2 AG/AG MSC and NRF2 +/+ MSC were used as test cells, respectively, and the amount of NRF2 protein in the test cells was detected.
  • the specific steps are as follows: RIPA cell lysate (Biyuntian, P0013B) was used to lyse the test cells, and then the BCA protein quantification kit (Biyuntian, P0010) was used to determine the protein concentration in the test cells, and finally 20 ug protein was used for Western blot.
  • the primary antibody was NRF2 (abcam, 62352) and the secondary antibody was goat anti-rabbit IgG/HRP (Zhongsu Jinqiao, ZDR-5306).
  • NRF2 AG/AG MSC and NRF2 +/+ MSC were used as test cells to detect NRF2 protein content in cytoplasm and nucleoplasm, respectively.
  • the specific steps are as follows: the cytoplasmic and nuclear component proteins were extracted using the nuclear protein and cytoplasmic protein extraction kit (Biyuntian, P0028), and then the same amount of protein was used for Western blot analysis, and the cytoplasmic marker ⁇ was used. -tubulin and the nucleoplasmic marker Lamin B1 were controls.
  • NRF2 AG/AG MSC and NRF2 +/+ MSC were used as test cells, respectively, and the RNA of the test cells was extracted and reverse transcribed into cDNA.
  • the following primers were used for real-time quantitative PCR to detect the stress-resistant gene NRF2 in the test cells.
  • Downstream target genes expression levels of NQO1 gene, HO-1 gene, GCLC gene, GCLM gene, GR gene, TXN gene, and TRXR1 gene.
  • NRF2 AG/AG MSC and NRF2 +/+ MSC were used as test cells, and the proteins in the test cells were extracted and subjected to Western blot to detect the expression levels of NQO1 and HO-1 in the cells.
  • the cytoplasmic marker ⁇ -tubulin was used as a control.
  • the antibody used was NQO1 (Santa Cruz Biotechnology, 32793), HO-1 (ENZO, ADI-SPA-895-D).
  • the NRF2 gene is constitutively activated after NRF2 single site modification.
  • test cells were treated with exogenous stimuli including oxidative stress, endoplasmic reticulum pressure, DNA damage stress, and apoptotic stimuli.
  • exogenous stimuli including oxidative stress, endoplasmic reticulum pressure, DNA damage stress, and apoptotic stimuli.
  • the specific steps are as follows: the test cells NRF2 AG/AG MSC and NRF2 +/+ MSC were plated on 96-well plates, and the exogenous stimuli (the final concentration of exogenous stimuli and the place of purchase in parentheses) were used to stimulate the supply.
  • Oxidative pressure DMSO (0.1%, Sigma), PQ (2 mM, Paraquat, Sigma), PX12 (50 uM, Santa Cruz Technology), TBH (100 uM, tert-Butyl hydroperoxide, Sigma), L-BSO (500 uM, L -Buthionine-sulfoximine, Sigma);
  • CCCP 100 uM, Carbonyl cyanide 3-chlorophenylhydrazone, Sigma.
  • the cell viability of the test cells after stimulation was measured according to the CellTiter 96AQueous One Solution Cell Proliferation Kit (Promega, G3582) kit and the method of use thereof.
  • CellTiter 96AQueous One Solution reagent was directly added to the culture medium of the cultured cells, and after 1 hour of incubation in a cell culture incubator, the absorbance value at 490 nm was measured, and the relative cell viability was calculated from the value of OD490.
  • the relative cell viability calculation formula is: Log2 (OD490(NRF2 AG/AG )/OD490(NRF2 +/+ )).
  • Cellular senescence ⁇ -galactosidase staining is a method for staining senescent cells or tissues based on up-regulation of SA-beta-Gal (senescence-associated beta-galactosidase) activity during senescence.
  • SA-beta-Gal senescence-associated beta-galactosidase
  • the NRF2 AG/AG MSC and NRF2 +/+ MSC obtained in the first step were stained with SA- ⁇ -Gal, and the senescence of the cells or tissues was observed under ordinary light microscope, and further in the two groups of cells.
  • the ratio of SA- ⁇ -Gal staining positive cells was quantitatively analyzed. Specific steps are as follows:
  • NRF2 AG/AG MSC and NRF2 +/+ MSC were used as test cells (early generation: 5th generation, late generation: 11th generation), washed once with PBS, and then added staining fixative (2% formaldehyde + 0.2) % glutaraldehyde), fixed at room temperature for 5 minutes. The fixative was discarded, washed once with PBS, and 1 ml of staining working solution was added to each well. Using X-Gal as a substrate, a dark blue product is formed under the catalysis of senescence-specific ⁇ -galactosidase.
  • Fig. 2C The results are shown in Fig. 2C. It can be seen from the figure that the late generation NRF2 +/+ MSC has obvious blue color, while the early generation NRF2 +/+ MSC and the late generation NRF2 AG/AG MSC (NRF2 enhanced MSC) The positive rate is very low. It can be seen that the aging process of NRF2 enhanced MSC is delayed.
  • NRF2 AG/AG MSC and NRF2 +/+ MSC were used as test cells (early generation EP: 5th generation, late generation LP: 11th generation), and 1 ⁇ M oxygen free radical probe DCFDA (Invitrogen, Cat. No. C6827) Incubate for 30 minutes and measure the DCFDA intensity (ie, the intracellular reactive oxygen species) by flow cytometry. Detailed steps can be found in the Invitrogen company instructions.
  • NRF2 AG/AG MSC NRF2 enhanced MSC
  • NRF2 enhanced MSC accumulated less active oxygen in the early and late generations, indicating that NRF2 enhanced MSC can more effectively remove this harmful metabolite.
  • NRF2 AG/AG MSC and NRF2 +/+ MSC were used as test cells (early generation EP: 5th generation, late generation LP: 11th generation), and Western blot analysis was used to detect senescence high expression genes p16 and p21 in the test cells. , GATA4 protein expression.
  • Primary antibodies were used as p16 (BD, 550834), P21 (Cell signaling, 2947), GATA4 (Santa cruz, SC-1237).
  • the corresponding secondary antibodies were goat anti-mouse IgG/HRP (Zhongsu Jinqiao, ZDR-5307), goat anti-rabbit IgG/HRP (Zhongsu Jinqiao, ZDR-5306) and rabbit anti-goat IgG/HRP (Zhongsu Jinqiao, ZDR-5308).
  • NRF2 AG/AG MSC and NRF2 +/+ MSC were used as test cells (early generation EP: 5th generation, late generation LP: 11th generation), and Ki67 expression related to proliferation activity was detected by immunofluorescence staining.
  • the specific steps are as follows: The test cells were plated on a circular slide, fixed with 4% paraformaldehyde, and permeabilized with 0.4% Triton X-100. The permeabilized cells were blocked in sputum serum for 1 h, followed by incubation with the primary antibody overnight, using the primary antibody Ki67 (Vector Laboratories, Cat. No. VP-RM04). Cell images were taken using a laser confocal microscope after incubation for the secondary antibody for 45 minutes.
  • NRF2 AG/AG MSC NRF2 enhanced MSC
  • Ki67 positive cell rate in both early and late generations than NRF2 +/+ MSC.
  • NRF2 AG/AG MSC and NRF2 +/+ MSC were used as test cells (early generation: 5th generation, late generation: 11th generation), and the expression of nuclear membrane proteins Lamin B1 and LAP2 were detected by immunofluorescence staining.
  • the specific steps are as follows: The test cells were plated on a circular slide, fixed with 4% paraformaldehyde, and permeabilized with 0.4% Triton X-100. The permeabilized cells were blocked in sputum serum for 1 h, and then the primary antibody was incubated overnight, using primary antibodies Lamin B1 (Abeam, Cat. No. 16048) and LAP2 (BD, Cat. No. 611000). Cell images were taken using a laser confocal microscope after incubation for the secondary antibody for 45 minutes.
  • the tumorigenic factors were transferred to NRF2 AG/AG MSC and NRF2 +/+ MSC, respectively, and the non-tumor MSCs were transformed into transformed MSCs (formerly called MSCs), which were respectively obtained by NRF2 AG. /AG TMSC and NRF2 +/+ TMSC. Tumor-related properties were subsequently detected. Specific steps are as follows:
  • the retroviral vector plasmids pBABE-neo-hTERT, pBABE-zeo-large T genomic, pBABE-puro-HRAS V12 required for the in vitro malignant transformation system were co-transfected with the packaging plasmids gag/pol and VSV-G to 293T, respectively.
  • the culture supernatant was collected, and the retrovirus particles were purified by ultracentrifugation.
  • the lentiviral vector plasmid PLVTHM-shP53 required for the in vitro malignant transformation system was co-transfected into the 293T cells with the packaging plasmids psPAX and pMD.2G, respectively, and the culture supernatant was collected, and the lentiviral particles were purified by ultracentrifugation.
  • the purified virus was infected with NRF2 AG/AG MSC and NRF2 +/+ MSC, respectively.
  • malignant transformation of MSCs includes telomere elongation, tumor suppressor gene deletion and oncogene mutation, which is achieved by infection with three retroviruses and one lentivirus.
  • the lentivirus can simultaneously express the green fluorescent GFP protein while achieving visual labeling of the cells.
  • the specific procedure was to sequentially infect the early generation MSCs with the pBABE-neo-hTERT, pBABE-zeo-large T genomic and pBABE-puro-HRAS V12 three retroviruses obtained in step (1).
  • the three viruses have the resistance markers of neo, zeo and puro, respectively, the corresponding G418 (Invitrogen, 10131035), zeocin (Invitrogen, R25001) and puromycin (Invitrogen, A1113803) were used for positive screening to obtain three.
  • the cells were integrated with the virus, and finally infected with PLVTHM-shP53 lentivirus enhanced the inhibition of the tumor suppressor gene and the labeling of GFP.
  • NRF2 AG/AG TMSC and NRF2 +/+ TMSC were obtained by this step.
  • NRF2 AG/AG TMSC and NRF2 +/+ TMSC were mixed with agarose (0.35%) in a dissolved state, and quickly spread in a cell culture dish. After agarose solidification, cover the TMSC medium and observe the non-anchor dependence. Growth ability.
  • NRF2 AG/AG TMSC and NRF2 +/+ TMSC (3 ⁇ 10 ⁇ 6 cells) were transplanted into the proximal joint of the hind limbs of nude mice (Balb/C nude mice).
  • the adherent cultured TMSC was digested into single cells, resuspended in a solution of PBS (Gibco, 10010023) mixed with 20% Matrigel (BD Biosciences, 354277), and the cell suspension was injected into the muscles of the hind limbs near the joint using a normal syringe. in. After 2-3 months of transplantation, when the hind limbs showed obvious swelling of the tumor, the mice were sacrificed.
  • NRF2-enhanced mesenchymal stem cells (NRF2-enhanced MSC) obtained in Example 2 have anti-reverse, anti-cell senescence and anti-tumor properties
  • the present invention uses the NRF2-enhanced mesenchymal stem cells obtained in Example 2. For cell transplantation treatment. Specific steps are as follows:
  • Luciferase labeling was performed on NRF2 AG/AG MSC and NRF2 +/+ MSC, respectively.
  • the specific procedure is as follows: a lentiviral vector plasmid overexpressing luciferase luciferase is co-transfected into 293T cells with lentiviral packaging plasmids psPAX and pMD2.G, the culture supernatant is collected, and the virus particles are purified by ultracentrifugation. The purified virus was infected with NRF2 AG/AG MSC and NRF2 +/+ MSC to obtain luciferase-labeled NRF2 AG/AG MSC and NRF2 +/+ MSC, respectively;
  • NRF2 AG/AG MSC and NRF2 +/+ MSC (1 ⁇ 10 ⁇ 6 cells) were transplanted into the tibialis anterior muscle of the mouse, and the animal retention image was used to detect the leg retention after 7 days. MSC.
  • NRF2 AG/AG MSC (NRF2 enhanced MSC) can survive longer than NRF2 +/+ MSC.
  • NRF2 AG/AG MSC and NRF2 +/+ MSC were transplanted into the muscles of immunodeficient mice with hindlimb ischemia, and the mice were detected by laser Doppler flowmetry on the 0th, 4th, 8th, 12th and 16th day after transplantation. Recovery of blood flow in the hind limbs.
  • a PBS solution Gibco, 10010023 was used as a control at the same time.
  • the invention successfully utilizes gene editing technology to successfully transform the stress-resistant related genes, and realizes the active activation of the endogenous stress resistance gene only by a single coding site modification, and obtains the gene-enhanced stem cells with improved stress resistance characteristics, and adopts in vivo and in vitro experiments. It is proved that gene-enhanced stem cells can resist stress and cell senescence, have better tissue repair function and the ability to resist malignant tumor formation.
  • the method of the invention simultaneously solves two key and bottleneck problems in the effectiveness and safety of cell transplantation therapy.

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Abstract

提供一种同时抵抗细胞衰老及恶性转化的人多能干细胞的制备方法。利用基因编辑技术将NRF2蛋白的第82位谷氨酸突变为甘氨酸，获得了抗逆特性提高的基因增强型干细胞，并通过体内体外实验证明了该干细胞能够抵抗逆境及细胞衰老，具有更佳的组织修复功能以及抵抗恶性成瘤转化的能力。

Description

一种同时抵抗细胞衰老及恶性转化的人多能干细胞的制备方法 技术领域
本发明属于生物技术领域，涉及一种同时抵抗细胞衰老及恶性转化的人多能干细胞的制备方法。
背景技术
细胞移植治疗是近年来再生医学领域最具前景和潜力的发展方向。细胞移植旨在补充或替代体内因疾病或衰老等因素导致的本已耗竭或失去正常功能的细胞，移植的细胞在体内存活并发挥正常功能，恢复组织内稳态，进而实现组织修复和再生。尽管细胞移植治疗已经在诸如骨髓干细胞移植治疗白血病等方面表达出极佳的治疗效果，但其更广泛的应用仍面临许多问题的挑战。目前，限制细胞移植治疗的主要因素集中在有效性和安全性两方面。
细胞移植治疗的有效性主要体现在移植物移植前的质量及进入体内后能否存活整合并发挥再生功能。一方面，移植物在体外培养过程中存在一定的“寿命”，具体表现为持续受到复制压力影响，有限的连续传代后迅速进入细胞老化(cellular senescence)状态，停止增殖且失去功能。另一方面，细胞移植的部位普遍位于疾病或衰老的组织微环境中，其中的炎性因子、有害代谢产物等会损害外源移植物。以上两方面都会导致细胞发生严重的功能性衰退，导致移植效率低下。细胞移植治疗的安全性主要集中在长期移植后的致瘤风险。细胞移植物在病体中持续受到压力胁迫，基因组可能变得极不稳定，若在癌基因或抑癌基因上发生突变，可能演变为癌症，形成安全隐患。
如何通过特定的干预手段提高细胞移植治疗的效率，尽可能的降低致瘤风险一直以来都是再生医学能够广泛应用的决定性问题。然而，迄今为止尚未发展出简单有效的方法克服这一技术瓶颈。当前提高细胞移植效果的尝试主要集中在改善移植部位微环境和增强移植物“抗逆”特性两方面。许多研究通过在不利细胞发挥正常功能的病体微环境中补充外源“营养及保护因子”的方式来改善移植效果，但该方法仅能在短期内改善移植环境，移植复发等不良预后问题突出。与此相比，通过特定方法改善移植物的“内因”，即增强移植材料自身固有的抵抗逆境能力来实现移植效果的改善是更为直接和长效的手段。目前已有少数研究表明可以利用慢病毒或逆转录病毒载体导入抗逆相关蛋白，调控细胞内特定的信号通路或基因表达，增强细胞对逆境的抵抗，进而实现细胞移植治疗效果的提升。尽管一系列临床前研究证明了提高细胞固有抗逆特性的可行性，但整合型病毒载体介导的基因调控必然存在基因组的随机整合，基因突变风险加大，安全性堪忧，这大大削弱了其在临床上的应用价值。
基因靶向编辑技术的发展极大的推动了细胞移植治疗的进步。这一技术使得人们可以精确的编辑基因组上的遗传密码，目前已有大量利用基因靶向编辑技术矫正疾病细胞致病突变的案例被报道。恢复正常功能的基因矫正细胞经体 外扩增可以用于自体移植，是细胞移植治疗中绝佳的材料。
发明公开
本发明的一个目的是提供一种同时抵抗细胞衰老及恶性转化的NRF2基因增强型的间充质干细胞的制备方法。
本发明提供的间充质干细胞的制备方法包括如下步骤：
(1)将离体的人多能干细胞中的NRF2蛋白的第82位的谷氨酸突变为甘氨酸，得到NRF2基因增强型的多能干细胞；
(2)对所述NRF2基因增强型的多能干细胞进行定向诱导分化，获得NRF2基因增强型的间充质干细胞。
上述方法中，步骤(1)中，所述将离体的人多能干细胞中的NRF2蛋白的第82位的谷氨酸突变为甘氨酸的方法为将NRF2基因的第245位的碱基A突变为碱基G；所述NRF2基因的第245位为NRF2基因编码区的第245位。
上述方法中，所述将NRF2基因的第245位的碱基A突变为碱基G的方法为基因组定点编辑。
上述方法中，所述基因组定点编辑的方法可为ZFN编辑、TALEN编辑、CRISPR/Cas9编辑或HdADV介导的定点编辑。
上述方法中，所述基因组定点编辑的方法为HdADV介导的定点编辑。
在本发明的实施例中，所述HdADV介导的定点编辑可通过如下方法实现：
第一步、突变片段的获得
将人类基因组NRF2基因第1和2号内含子(intron1-exon2-intron2)的基因片段中对应NRF2基因的cDNA序列的第245位的核苷酸位点，由野生型的碱基A突变为碱基G，且保持其他序列不变，得到突变后的片段；
第二步、病毒表达质粒的构建
将所述突变后的片段插入pCIHDAdGT8-4载体的AscI和SpeI酶切位点中，得到重组质粒。
第三步、病毒表达质粒与辅助质粒共转染包装细胞获得重组腺病毒颗粒
用PI-SceI(NEB)酶切所述重组质粒，得到线性化质粒，并将所述线性化质粒同辅助病毒AdHPBGF35共同导入包装细胞中进行重组腺病毒包装，得到重组腺病毒颗粒；所述包装细胞具体为人胚胎肾细胞293系的衍生细胞系116细胞；
第四步、重组腺病毒颗粒感染目的细胞
用重组腺病毒颗粒感染人多能干细胞，经过筛选，得到一个NRF2等位基因上位点正确编辑的细胞，将其记作NRF2AG/+hESC细胞；
第五步、将所述线性化质粒和所述辅助病毒AdHPBGF35共同导入所述NRF2AG/+hESC细胞中再次进行基因编辑，并对编辑后细胞进行鉴定，获得两个NRF2等位基因上位点正确编辑的细胞，即为NRF2基因增强型的多能干细胞。
上述方法中，步骤(2)中，所述定向诱导分化的方法包括如下步骤：
(b1)将NRF2基因增强型的多能干细胞进行拟胚体分化，获得拟胚体；
(b2)培养所述拟胚体至纤维状细胞出现；再经过传代培养，分选其中CD73、CD90、CD105均为阳性的细胞类群，即为所述NRF2基因增强型的间充质干细胞。
上述方法中，所述拟胚体分化的具体方法如下：准备含有300-500个细胞、大小均一的NRF2AG/AG hESC克隆，用室温PBS清洗一次，再用Dispase 37℃消化20-30min。待ESC克隆形成球体后，用CDF12培养基重悬，然后加到低粘附培养板(Corning公司，货号3471)中，37℃，5％CO2条件培养1-3天后即形成拟胚体。
培养所述拟胚体至纤维状细胞出现的具体方法如下：将所述拟胚体接种于基质胶包被的6孔板中进行培养，继续培养2周至纤维状细胞出现。
上述方法中，所述人多能干细胞为人胚胎干细胞或人诱导多能干细胞。
上述方法中，所述人胚胎干细胞具体为人胚胎干细胞H9细胞系。
本发明的另一个目的是提供利用上述方法制备获得的NRF2基因增强型的间充质干细胞。
利用上述方法制备获得的NRF2基因增强型的多能干细胞也属于本发明的保护范围。
本发明还有一个目的是提供上述NRF2基因增强型的间充质干细胞或上述NRF2基因增强型的多能干细胞的新用途。
本发明提供了上述NRF2基因增强型的间充质干细胞或上述NRF2基因增强型的多能干细胞在如下c1)-c5)中任一种中的应用：
c1)抵抗和/或延缓细胞衰老；
c2)抵抗肿瘤发生；
c3)抵抗恶性成瘤转化；
c4)细胞移植治疗；
c5)再生和/或修复损伤血管。
本发明还提供了上述NRF2基因增强型的间充质干细胞或上述NRF2基因增强型的多能干细胞在如下d1)-d5)中任一种中的应用：
d1)制备抵抗和/或延缓细胞衰老的产品；
d2)制备抵抗肿瘤发生的产品；
d3)制备抵抗恶性成瘤转化的产品；
d4)制备细胞移植治疗的产品；
d5)制备再生和/或修复损伤血管的产品。
本发明最后一个目的提供一种产品。
本发明提供的产品的活性成分为上述NRF2基因增强型的间充质干细胞；
所述产品的功能为如下m1)-m5)中任一种：
m1)抵抗和/或延缓细胞衰老；
m2)抵抗肿瘤发生；
m3)抵抗恶性成瘤转化；
m4)细胞移植治疗；
m5)损伤血管的再生和/或修复。
上述应用或上述产品中，
所述抵抗和/或延缓细胞衰老体现在如下n1)-n6)中任一种：
n1)在外源刺激物刺激下细胞活力增强；
n2)在体外连续传代培养过程中表现更强的增殖能力；
n3)清除有害代谢物的能力增强；
n4)与细胞衰老相关基因的表达水平降低；所述与细胞衰老相关基因具体为p16基因、p21基因和GATA4基因；
n5)具有更完整的细胞核结构；
n6)在人或动物体内存留时间更长。
上述应用或上述产品中，
所述抵抗恶性成瘤转化体现在如下e1)-e3)中的任一种：
e1)瘤化细胞增殖能力降低；
e2)与肿瘤形成相关的非锚定依赖的生长能力降低；
e3)在体内形成肿瘤的能力降低；
上述应用或上述产品中，所述细胞移植治疗为细胞移植修复治疗。
附图说明
图1为本发明产生了抗逆基因NRF2单一位点编辑的多能干细胞，并由此衍生出基因增强型间充质干细胞，此细胞中的NRF2活性得到持续激活。其中，A为基因编辑的模式图；B为NRF2基因A245G位点的测序结果；C为获得的多能干细胞的形态及体内三胚层分化的免疫荧光鉴定；D为多能干细胞中干性基因的表达情况；E为分化得到的MSC中与MSC有关和无关的表面标记物鉴定情况；F为MSC中NRF2蛋白在细胞核内外的含量分析；G为NRF2下游靶基因的mRNA水平；H为NRF2及其关键下游靶基因NQO1和HO-1的蛋白表达情况。
图2为基因增强型间充质干细胞抵抗逆境及细胞衰老的特性。其中，A为外源刺激物处理下细胞活力检测；B为MSC生长曲线；C为SA-β-Gal染色结果；D为活性氧含量检测；E为衰老相关标记物表达情况的结果；F为细胞增殖相关标记物Ki67的检测；G为核膜完整性检测。
图3为基因增强型间充质干细胞抵抗恶性转化的特性。其中，A为体外恶性转化模式图；B为恶性转化后MSC非锚定依赖的生长情况；C为恶性转化后MSC的体内成瘤能力；D为小鼠长瘤部分的横纵向切片染色鉴定。
图4为移植基因增强型间充质干细胞应用于后肢缺血模型血流恢复的实例。其中，A为MSC在肌肉微环境中的留存能力；B为每只后肢缺血小鼠血流恢复情况；C为不同时间点小鼠腿部的代表性图片。
实施发明的最佳方式
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
下述实施例中的定量试验，均设置三次重复实验，结果取平均值。
1、下述实施例中的培养基配方如下：
(1)CDF12培养基配方：
DMEM/F12培养基(Invitrogen，11320-033)；
0.1mM非必需氨基酸(Invitrogen，11140-050)；
1mM GlutaMAX(Invitrogen，35050-061)；
20％(体积百分含量)Knockout血清替代物(Invitrogen，N10828-028)；
1％(1g/100ml)青霉素/链霉素(Invitrogen，15070-063)；
55μMβ-巯基乙醇(Invitrogen，21985-023)；
10ng/ml人FGF2(Joint Protein Central)。
(2)间充质干细胞(MSC)培养基配方：
MEM培养基(Invitrogen，12571071)；
10％(体积百分含量)胎牛血清(Invitrogen，10091148)；
1％(1g/100ml)青霉素/链霉素(Invitrogen，15070-063)；
10ng/ml重组人成纤维细胞生长因子(JPC，bFGF)；
MSC分化培养基需额外添加5ng/ml TGFβ(Humanzyme，HZ1131)。
2、细胞系如下：
人胚胎干细胞H9细胞系是WiCell公司产品，货号：WA09(H9)-DL-7。
人胚胎肾细胞293T系购自ATCC，货号CRL-3216。
3、人胚胎干细胞H9细胞系培养方法如下：
(1)将H9细胞接种至预先培养了经过丝裂霉素(美国Sigma公司产品，货号：M0503)灭活的小鼠胚胎成纤维细胞(美国Invitrogen公司产品，货号：S1520-100)的培养板中，使用人类胚胎干细胞培养基(CDF12培养基)与小鼠胚胎成纤维细胞共同培养；
(2)将H9细胞接种至预先用细胞外基质(qualified-Matrigel，美国BD Biosciences产品，货号：354277)包被的培养板中，使用mTeSR培养基(美国StemCell Technologies产品)培养。
4、病毒包装所用生物材料如下：
(1)重组腺病毒包装所用生物材料如下：
pCR2.1-TOPO载体为Invitrogen公司产品。
辅助腺病毒载体pCIHDAdGT8-4载体：详见“An HSV amplicon-based helper system for helper-dependent adenoviral vectors.Shuji Kubo,et al.BBRC.2003.307(4):826-830”一文，公众可从原文作者或中国科学院生物物理研究所获得。
转染人胚胎肾细胞293系的衍生细胞系116细胞：详见“Improved system  for helper-dependent adenoviral vector production.Palmer D.and Ng P.Molecular Therapy.2003.8(5):846-52.”一文，公众可从原文作者或中国科学院生物物理研究所获得。
辅助腺病毒AdHPBGF35：详见“Genome Size and Structure Determine Efficiency of Postinternalization Steps and Gene Transfer of Capsid-Modified Adenovirus Vectors in a Cell-Type-Specific Manner.Dmitry M.Shayakhmetov,et al.,Journal of Virology.2004.78(18):10009-10022.”一文，公众可从原文作者或中国科学院生物物理研究所获得。
(2)逆转录病毒包装所用生物材料如下：
逆转录病毒载体及包装质粒均购自Addgene，货号如下：pBABE-neo-hTERT(1774)，pBABE-zeo-large T genomic(1778)，pBABE-puro-HRAS V12(1768)，gag/pol(14887)，VSV-G(8454)。
(3)慢病毒包装所用生物材料如下：
过表达荧光素酶luciferase的慢病毒载体质粒：详见“SIRT6safeguards human mesenchymal stem cells from oxidative stress by coactivating NRF2.Pan et.al,.Cell research.(2016)26:190-205.”一文，公众可从中国科学院生物物理研究所获得。
P53敲低的慢病毒载体质粒PLVTHM-shP53：详见“PTEN deficiency reprogrammes human neural stem cells towards a glioblastoma stem cell-like phenotype.Duan et.al,.Nature communication.(2015)10.1038.”一文，公众可从中国科学院生物物理研究所获得。
慢病毒包装质粒购自Addgene，货号如下：psPAX(12260)，pMD2.G(12259)。
5、用于流式细胞术分选MSC的荧光标记抗体如下：
荧光素FITC标记的抗人细胞表面识别分子CD90抗体，BD Biosciences，货号：555595。
荧光素PE标记的抗人细胞表面识别分子CD73抗体，BD Biosciences，货号：550257。
荧光素APC标记的抗人细胞表面识别分子CD105抗体，BD Biosciences，货号：17-1057-42。
荧光素APC标记同型对照抗体，BD Biosciences，货号：555751。
荧光素PE标记同型对照抗体，BD Biosciences，货号：555749。
荧光素FITC标记同型对照抗体，BD Biosciences，货号：555742。
6、用于免疫荧光的抗体如下：
抗人FOXA2抗体，Cell Signaling Technology，货号：8186S。
抗人SMA抗体，中杉金桥，货号：ZM-0003。
抗人TUJ1抗体，Sigma，货号：T2200。
抗人LAP2抗体，BD Biosciences，货号：611000。
抗人Lamin B1抗体，Santa Cruz Biotechnology，货号：sc-6217。
抗人Ki 67抗体，Vector Laboratories，货号：VP-RM04。
抗人P16抗体，BD Biosciences，货号：550834。
抗人P21抗体，Cell Signaling Technology，货号：2947。
实施例1、抗逆基因NRF2靶向修饰的人类多能干细胞的制备
一、NRF2增强型多能干细胞的制备
本发明通过对多能干细胞(Pluripotent Stem Cell,PSC)中的人类抗逆基因NRF2的单一位点进行靶向编辑，使NRF2基因(GeneBank ID：4780)的cDNA序列的第245位由野生型的碱基A突变为碱基G，NRF2蛋白的氨基酸序列的第82位由谷氨酸(E)变为甘氨酸(G)，获得NRF2增强型多能干细胞。具体步骤如下(具体步骤参见图1A)：
1、病毒表达质粒的构建
首先从RP11-483K11BAC DNA文库(BACPAC Resources)中通过PCR扩增获得跨人类基因组NRF2基因第1和2号内含子(intron1-exon2-intron2)的基因片段。然后通过定点突变试剂盒(Invitrogen，货号：A13282)突变该片段上对应NRF2基因的cDNA序列的第245位的核苷酸位点(A245G)，由野生型的碱基A突变为碱基G，且保持其他序列不变，得到突变后的片段，并将突变后的片段插入pCIHDAdGT8-4载体的AscI和SpeI酶切位点中，得到重组质粒。
2、病毒表达质粒与辅助质粒共转染包装细胞获得重组腺病毒颗粒
用PI-SceI(NEB)酶切步骤1获得的重组质粒，得到线性化质粒，并将该线性化质粒同辅助病毒AdHPBGF35共同导入人胚胎肾细胞293系的衍生细胞系116细胞中进行重组腺病毒包装，并通过超高速离心纯化获得重组腺病毒颗粒。
3、重组腺病毒颗粒感染目的细胞
用重组腺病毒颗粒感染hESCs细胞(H9)(1×107个细胞，病毒用量为15bgal-transducing units(btu)，病毒用量单位可参见文献“Palmer,D.J.&Ng,P.Physical and infectious titers of helper-dependent adenoviral vectors:a method of direct comparison to the adenovirus reference material.Mol Ther 10,792‐798(2004).”和“Suzuki,K.et al.Highly efficient transient gene expression and gene targeting in primate embryonic stem cells with helper-dependent adenoviral vectors.Proc Natl Acad Sci U S A 105,13781‐13786(2008).”，感染后第2-4天，加入G418(25～400g/ml，invitrogen公司产品)进行阳性的筛选，感染后第10-13天，加入4μM Ganciclovir(GANC,invitrogen公司产品)进行阴性的筛选。最后对存活下来的细胞进行测序鉴定(PCR引物为NRF2A245GseqF：ACCATTTGTGACTTTGCCCTTTAGTGACCTCTACCATC和NRF2A245GseqR：AACCTGCCATAACTTTCCCAAGAACTGA，测序引物为aaaaacagaaaaaacttgaa)，得到 一个NRF2等位基因上位点正确编辑的细胞，将其记作NRF2AG/+hESC细胞。
4、将上述线性化质粒同辅助病毒AdHPBGF35共同导入NRF2AG/+hESC细胞中再次进行基因编辑，并对编辑后细胞进行鉴定，获得两个NRF2等位基因上位点正确编辑的细胞，将其记作NRF2AG/AG hESC细胞，即NRF2增强型多能干细胞。
二、NRF2增强型多能干细胞的鉴定
1、NRF2增强型多能干细胞的测序鉴定
对NRF2增强型多能干细胞进行测序鉴定。经测序鉴定表明：NRF2增强型多能干细胞中NRF2基因的A245G位点发生正确编辑(图1B)，NRF2增强型成体细胞中的NRF2基因的两条同源染色体的第245位均由碱基A突变为碱基G。
2、NRF2增强型多能干细胞的形态鉴定
对NRF2增强型多能干细胞进行形态鉴定，NRF2AG/AG hESC的形态鉴定结果如图1C所示。此外从图中可以看出，NRF2增强型多能细胞依然具有在体内分化为内中外三胚层细胞的能力。
3、NRF2增强型多能干细胞中多能干性基因的表达水平检测
分别对野生型人胚胎干细胞H9细胞系(NRF2+/+)、NRF2AG/+hESC细胞(NRF2AG/+hESC)和NRF2AG/AG hESC细胞(NRF2AG/AG hESC)的表达多能干性基因OCT4、SOX2、NANOG的表达水平进行检测。用于PCR鉴定的引物如下：
Figure PCTCN2017080393-appb-000001
结果如图1D所示。从图中可以看出：NRF2+/+、NRF2AG/+hESC和NRF2AG/AG hESC细胞均正常表达多能干性基因OCT4、SOX2、NANOG。
实施例2、NRF2增强型间充质干细胞的制备及其功能检测
本发明将实施例1中获得的NRF2增强型多能干细胞进一步分化为NRF2增强型间充质干细胞(NRF2增强型MSC)。并通过实验证明：NRF2增强型MSC中NRF2得到主动激活，在体内体外均表现出抗逆、抗细胞衰老和抵抗恶性转化的特性。具体步骤如下：
一、NRF2增强型MSC的制备
1、将NRF2AG/AG hESC进行拟胚体(EB)分化，具体步骤如下：准备含有300-500个细胞、大小均一的NRF2AG/AG hESC克隆，用室温PBS清洗一次，用Dispase(Invitrogen公司，货号为17105041)37℃消化20-30min。待ESC克隆形成 球体后，用CDF12培养基重悬后，加到低粘附培养板(Corning公司，货号3471)中，37℃，5％CO2条件培养1-3天后即形成拟胚体。
2、将步骤1获得的拟胚体接种于基质胶(matrigel)(Invitrogen公司)包被的6孔板中进行培养，继续培养2周至纤维状细胞出现。再经过一次传代后，利用流式细胞术分选其中的CD73、CD90和CD105均为阳性的细胞类群，即为NRF2增强型MSC，将其记作NRF2AG/AG MSC。
将上述步骤中的NRF2AG/AG hESC替换为野生型人胚胎干细胞H9细胞系，其他步骤不变，定向诱导分化得到野生型间充质细胞记作NRF2+/+MSC。
二、NRF2增强型MSC的表型鉴定及功能检测
1、NRF2增强型MSC的表型鉴定
对步骤一获得的NRF2AG/AG MSC和NRF2+/+MSC的表面标记物进行检测。
结果如图1E所示。从图中可以看出：步骤一获得的NRF2AG/AG MSC和NRF2+/+MSC均能够表达MSC特异的表面标记物CD73，CD90和CD105，而不表达无关的CD34，CD43和CD45。
2、NRF2增强型MSC中抗逆基因NRF2的主动激活
(1)NRF2增强型MSC中NRF2蛋白量
分别以NRF2AG/AG MSC和NRF2+/+MSC为供试细胞，检测供试细胞中NRF2蛋白量。具体步骤如下：使用RIPA细胞裂解液(碧云天，P0013B)裂解供试细胞，然后使用BCA蛋白定量试剂盒(碧云天，P0010)测定供试细胞中蛋白浓度，最后分别取20ug蛋白进行Western blot实验。使用一抗为NRF2(abcam，62352)，二抗为羊抗兔IgG/HRP(中杉金桥，ZDR-5306)。
结果如图1H所示。从图中可以看出：NRF2AG/AG MSC(NRF2增强型MSC)中NRF2蛋白量明显高于NRF2+/+MSC。
(2)核内定位
分别以NRF2AG/AG MSC和NRF2+/+MSC为供试细胞，分别检测胞质及核质中NRF2蛋白含量。具体步骤如下：使用细胞核蛋白与细胞浆蛋白抽提试剂盒(碧云天，P0028)分别提取胞质及核质组分蛋白，然后取等量蛋白分别进行Western blot实验，并以胞质标记物β-tubulin和核质标记物Lamin B1为对照。
结果如图1F所示。从图中可以看出：NRF2AG/AG MSC(NRF2增强型MSC)中的NRF2蛋白在胞质及核质中的含量均高于NRF2+/+MSC。
(3)NRF2增强型MSC中抗逆基因NRF2下游靶基因的mRNA和蛋白水平检测
1)实时定量PCR实验
分别以NRF2AG/AG MSC和NRF2+/+MSC为供试细胞，分别提取供试细胞的RNA并反转录为cDNA，分别使用如下引物进行实时定量PCR，检测供试细胞中抗逆基因NRF2下游靶基因：NQO1基因、HO-1基因、GCLC基因、GCLM基因、GR基因、TXN基因、TRXR1基因的表达量。
Figure PCTCN2017080393-appb-000002
Figure PCTCN2017080393-appb-000003
2)分别以NRF2AG/AG MSC和NRF2+/+MSC为供试细胞，提取供试细胞中的蛋白并进行Western blot实验，检测供试细胞中NQO1和HO-1蛋白表达水平。并以胞质标记物β-tubulin为对照。使用抗体为NQO1(Santa Cruz Biotechnology，32793)，HO-1(ENZO，ADI-SPA-895-D)。
结果如图1G和图1H所示。从图中可以看出：NRF2AG/AG MSC(NRF2增强型MSC)中下游靶基因：NQO1基因、HO-1基因、GCLC基因、GCLM基因、GR基因、TXN基因和TRXR1基因的表达量均高于NRF2+/+MSC。且NRF2AG/AG MSC中NQO1和HO-1蛋白表达水平也高于NRF2+/+MSC。
综上所述，NRF2单一位点修饰后NRF2基因得到组成型激活。
3、NRF2增强型MSC的抗逆抗细胞衰老特性
(1)外源刺激下的高活力
使用包括氧化压力，内质网压力，DNA损伤压力及凋亡刺激在内的外源刺激物处理MSC。具体步骤如下：将供试细胞NRF2AG/AG MSC和NRF2+/+MSC铺于96孔板上，分别使用如下外源刺激物(括号内为外源刺激物的终浓度及购买处)刺激供试细胞48小时：
1)氧化压力：DMSO(0.1％，Sigma)、PQ(2mM，Paraquat，Sigma)、PX12(50uM，Santa Cruz Technology)、TBH(100uM，tert-Butyl hydroperoxide，Sigma)、L-BSO(500uM，L-Buthionine-sulfoximine，Sigma)；
2)内质网压力：TM(6ug/mL，Tunicamycin，Sigma)和TG(1.5ug/mL，Thapsigargin，Sigma)；
3)DNA损伤：4NQO(100uM，4-nitroquinoline N-oxide,Sigma)和AAT2(50uM，Apoptosis Activator 2,TOCRIS)；
4)凋亡：CCCP(100uM,Carbonyl cyanide 3-chlorophenylhydrazone,Sigma)。
刺激结束后按CellTiter 96AQueous One Solution Cell Proliferation Kit(Promega,G3582)试剂盒及其使用方法检测刺激后供试细胞的活力。检测时直接向培养细胞的培养基中加入CellTiter 96AQueous One Solution试剂，细胞培养箱中孵育1小时后检测490nm的吸光度值，根据OD490的数值计算相对细胞活力。相对细胞活力计算公式为：Log2(OD490(NRF2AG/AG)/OD490(NRF2+/+))。
结果表明：在本底水平及刺激条件下，NRF2增强型MSC均具有更强的细胞活力(图2A)。
(2)生长能力测定
利用细胞计数统计连续传代培养的NRF2AG/AG MSC和NRF2+/+MSC的生长能力。
结果如图2B所示。从图中可以看出：NRF2AG/AG MSC细胞(NRF2增强型MSC)倍增速率明显高于NRF2+/+MSC细胞，表现出明显的抵抗细胞衰老的能力。
(3)SA-β-Gal染色
细胞衰老β-半乳糖苷酶染色是一种基于衰老时SA-beta-Gal(senescence-associated beta-galactosidase)活性水平上调而对衰老细胞或组织进行染色检测的方法。分别对步骤一获得的NRF2AG/AG MSC和NRF2+/+MSC进行SA-β-Gal染色，并在普通的光学显微镜下就可以观测到细胞或组织的衰老情况，并进一步对两组细胞中的SA-β-Gal染色阳性细胞比率进行定量统计分析。具体步骤如下：
分别以NRF2AG/AG MSC和NRF2+/+MSC为供试细胞(早代：第5代，晚代：第11代)，用PBS洗涤1次，再加入染色固定液(2％甲醛+0.2％戊二醛)，室温固定5分钟。弃去固定液，用PBS洗涤1次，每孔加入1ml染色工作液。以X-Gal为底物，在衰老特异性的β-半乳糖苷酶催化下会生成深蓝色产物。
结果如图2C所示，从图中可以看出，晚代的NRF2+/+MSC有明显的蓝色，而早代NRF2+/+MSC及晚代的NRF2AG/AG MSC(NRF2增强型MSC)阳性率很低。可见，NRF2增强型MSC的衰老进程延缓。
(4)有害代谢物含量检测
分别以NRF2AG/AG MSC和NRF2+/+MSC为供试细胞(早代EP：第5代，晚代LP：第11代)，与1μM氧自由基探针DCFDA(Invitrogen，货号：C6827)共同孵育30分钟，利用流式细胞仪检测DCFDA强度(即指针细胞内活性氧含量)。详细步骤可参考Invitrogen公司说明书。
结果如图2D所示，NRF2AG/AG MSC(NRF2增强型MSC)在早晚代中都积累更少的活性氧，表明NRF2增强型MSC能够更有效的清除此有害代谢物。
(5)衰老基因表达检测
分别以NRF2AG/AG MSC和NRF2+/+MSC为供试细胞(早代EP：第5代，晚代LP：第11代)，Western blot实验检测供试细胞中衰老高表达基因p16、p21、GATA4的蛋白表达情况。使用一抗为p16(BD,550834)，P21(Cell signaling,2947)， GATA4(Santa cruz,SC-1237)。对应二抗为羊抗小鼠IgG/HRP(中杉金桥，ZDR-5307)，羊抗兔IgG/HRP(中杉金桥，ZDR-5306)及兔抗羊IgG/HRP(中杉金桥，ZDR-5308)。
结果如图2E所示。从图中可以看出，和NRF2+/+MSC相比，基因增强型MSC(NRF2AG/AG MSC)始终表达低丰度的衰老相关基因p16、p21和GATA4。
(6)增殖能力检测
分别以NRF2AG/AG MSC和NRF2+/+MSC为供试细胞(早代EP：第5代，晚代LP：第11代)，通过免疫荧光染色法检测与增殖活力相关的Ki67表达。具体步骤如下：供试细胞铺于圆形玻片上，使用4％多聚甲醛固定后经0.4％TritonX-100通透。通透的细胞在驴血清中封闭1h，随后一抗孵育过夜，使用一抗为Ki67(Vector Laboratories，货号：VP-RM04)。对应二抗孵育45分钟后使用激光共聚焦显微镜拍摄细胞图片。
结果如图2F所示。从图中可以看出，和NRF2+/+MSC相比，NRF2AG/AG MSC(NRF2增强型MSC)在早晚代中均具有更高的Ki67阳性细胞率。
(7)细胞核结构完整性检测
分别以NRF2AG/AG MSC和NRF2+/+MSC为供试细胞(早代：第5代，晚代：第11代)，通过免疫荧光染色法检测核膜蛋白Lamin B1和LAP2的表达。具体步骤如下：供试细胞铺于圆形玻片上，使用4％多聚甲醛固定后经0.4％TritonX-100通透。通透的细胞在驴血清中封闭1h，随后一抗孵育过夜，使用一抗为LaminB1(Abcam，货号：16048)及LAP2(BD，货号：611000)。对应二抗孵育45分钟后使用激光共聚焦显微镜拍摄细胞图片。
结果如图2G所示。从图中可以看出：和NRF2+/+MSC相比，晚代的NRF2AG/AG MSC(NRF2增强型MSC)核膜异常的细胞(图2G中箭头所指的即为核膜异常细胞，核膜蛋白染色后不可见明显的核膜轮廓)数目明显减少，表明NRF2AG/AG MSC(NRF2增强型MSC)具有更完整的细胞核结构。
4、NRF2增强型MSC抵抗恶性转化的特性
(1)NRF2AG/AG TMSC和NRF2+/+TMSC的制备
利用体外恶性转化系统，分别向NRF2AG/AG MSC和NRF2+/+MSC中转入致瘤因子，将非瘤的MSC转化为瘤化的MSC(transformed MSC，下称TMSC)，分别得到NRF2AG/AG TMSC和NRF2+/+TMSC。随后检测肿瘤相关特性。具体步骤如下：
1)将体外恶性转化系统所需逆转录病毒载体质粒pBABE-neo-hTERT、pBABE-zeo-large T genomic、pBABE-puro-HRAS V12分别与包装质粒gag/pol和VSV-G共转染至293T细胞中，收集培养上清，并通过超高速离心纯化逆转录病毒颗粒。将体外恶性转化系统所需慢病毒载体质粒PLVTHM-shP53分别与包装质粒psPAX和pMD.2G共转染至293T细胞中，收集培养上清，并通过超高速离心纯化慢病毒颗粒。
2)利用纯化得到的病毒分别感染NRF2AG/AG MSC和NRF2+/+MSC。如图3A所示， MSC的恶性转化包括端粒延长，抑癌基因缺失和致癌基因突变三方面，该过程通过感染3种逆转录病毒和1种慢病毒实现。慢病毒可同时表达绿色荧光GFP蛋白，同时实现细胞的可视标记。具体操作过程为在早代MSC中依次感染步骤(1)中获得的pBABE-neo-hTERT、pBABE-zeo-large T genomic以及pBABE-puro-HRAS V12三种逆转录病毒。由于三种病毒分别具有neo，zeo和puro的抗性标记，因而依次使用对应的G418(Invitrogen,10131035)，zeocin(Invitrogen,R25001)以及puromycin(Invitrogen,A1113803)三种药物进行正向筛选获得三种病毒均整合的细胞，最后感染PLVTHM-shP53慢病毒加强抑癌基因的抑制及GFP的标记。经此步骤分别得到NRF2AG/AG TMSC和NRF2+/+TMSC。
(2)非锚定依赖生长能力
肿瘤细胞在无法粘附的琼脂上能够长成克隆，而非瘤细胞则不可，因此非锚定依赖的生长能力与肿瘤细胞形成实体肿瘤密切相关。分别将NRF2AG/AG TMSC和NRF2+/+TMSC与溶解状态的琼脂糖(0.35％)混合均匀，迅速铺于细胞培养皿中，待琼脂糖凝固后覆盖TMSC培养基培养，观察非锚定依赖生长能力。
结果如图3B所示：NRF2AG/AG TMSC经恶性转化后其非锚定依赖生长能力较野生型细胞转化后大大减弱(图3B)。
(3)体内成瘤能力
分别将NRF2AG/AG TMSC和NRF2+/+TMSC(3×10^6个细胞)移植入裸鼠(品系：Balb/C nude mice)后肢近关节处。将贴壁培养的TMSC消化为单细胞，重悬于混有20％Matrigel(BD Biosciences，354277)的PBS(Gibco，10010023)溶液中，使用普通注射器将细胞悬液注射入后肢近关节处的肌肉中。移植2-3月后，当后肢出现明显肿物隆起，处死小鼠。切下后肢使用天平称重，并计算相对质量(相对质量＝移植NRF2+/+TMSC的后肢质量/移植NRF2AG/AG TMSC的后肢质量)。
结果如图3C和图3D所示：从图中可以看出，相对质量为2.73±0.22，移植NRF2+/+TMSC的后肢质量显著大于移植NRF2AG/AG TMSC的后肢质量。说明NRF2+/+TMSC能够长出骨肉瘤状肿瘤，而NRF2AG/AG TMSC则彻底丧失了形成肿瘤的能力。
实施例3、NRF2增强型间充质干细胞在细胞移植治疗中的应用
由于实施例2获得的NRF2增强型间充质干细胞(NRF2增强型MSC)具有抗逆、抗细胞衰老及抵抗肿瘤发生的特性，本发明将实施例2中获得的NRF2增强型间充质干细胞用于细胞移植治疗。具体步骤如下：
一、将NRF2增强型MSC移植到小鼠的胫骨前肌
1、分别对NRF2AG/AG MSC和NRF2+/+MSC进行荧光素酶标记。具体步骤如下：将过表达荧光素酶luciferase的慢病毒载体质粒与慢病毒包装质粒psPAX和pMD2.G共转染至293T细胞中，收集培养上清，并通过超高速离心纯化病毒颗粒。利用纯化得到的病毒感染NRF2AG/AG MSC和NRF2+/+MSC，分别得到荧光素酶标记的 NRF2AG/AG MSC和NRF2+/+MSC；
2、分别将荧光素酶标记的NRF2AG/AG MSC和NRF2+/+MSC(1×10^6个细胞)移植到小鼠的胫骨前肌中，7天后利用动物活体成像仪检测腿部存留的MSC。
结果如图4A所示，NRF2AG/AG MSC(NRF2增强型MSC)较NRF2+/+MSC能够存留更久。
二、将NRF2增强型MSC移植到后肢缺血的免疫缺陷小鼠肌肉
分别将NRF2AG/AG MSC和NRF2+/+MSC移植到后肢缺血的免疫缺陷小鼠肌肉中，利用激光多普勒血流仪检测移植后第0，4，8，12，16天小鼠后肢血流的恢复情况。同时将PBS溶液(control)(Gibco，10010023)作为对照。
结果如图4B和4C所示，较注射PBS溶液或移植NRF2+/+MSC相比，移植NRF2AG/AG MSC(NRF2增强型MSC)能够更快的恢复受损后肢的血流，表明NRF2AG/AG MSC参与了损伤后血管的再生和修复。
工业应用
本发明首次利用基因编辑技术成功改造抗逆相关基因，仅单一编码位点修饰便实现内源抗逆基因可控的主动激活，获得了抗逆特性提高的基因增强型干细胞，并通过体内体外实验证明了基因增强型干细胞能够抵抗逆境及细胞衰老，具有更佳的组织修复功能以及抵抗恶性成瘤转化的能力。本发明的方法同时解决了细胞移植治疗中有效性与安全性两大关键和瓶颈性难题。

Claims (16)

  1. 一种间充质干细胞的制备方法，包括如下步骤：
    (1)将离体的人多能干细胞中的NRF2蛋白的第82位的谷氨酸突变为甘氨酸，得到NRF2基因增强型的多能干细胞；
    (2)对所述NRF2基因增强型的多能干细胞进行定向诱导分化，获得NRF2基因增强型的间充质干细胞。
  2. 根据权利要求1所述的方法，其特征在于：步骤(1)中，所述将离体的人多能干细胞中的NRF2蛋白的第82位的谷氨酸突变为甘氨酸的方法为将NRF2基因的第245位的碱基A突变为碱基G。
  3. 根据权利要求2所述的方法，其特征在于：所述将NRF2基因的第245位的碱基A突变为碱基G的方法为基因组定点编辑。
  4. 根据权利要求3所述的方法，其特征在于：
    所述基因组定点编辑的方法可为ZFN编辑、TALEN编辑、CRISPR/Cas9编辑或HdADV介导的定点编辑。
  5. 根据权利要求4所述的方法，其特征在于：
    所述基因组定点编辑的方法为HdADV介导的定点编辑。
  6. 根据权利要求1所述的方法，其特征在于：
    步骤(2)中，所述定向诱导分化的方法包括如下步骤：
    (b1)将NRF2基因增强型的多能干细胞进行拟胚体分化，获得拟胚体；
    (b2)培养所述拟胚体至纤维状细胞出现；再经过传代培养，分选其中CD73、CD90、CD105均为阳性的细胞类群，即为所述NRF2基因增强型的间充质干细胞。
  7. 根据权利要求6所述的方法，其特征在于：
    所述人多能干细胞为人胚胎干细胞或人诱导多能干细胞。
  8. 根据权利要求7所述的方法，其特征在于：
    所述人胚胎干细胞为人胚胎干细胞H9细胞系。
  9. 利用权利要求1-8中任一所述方法制备获得的NRF2基因增强型的间充质干细胞。
  10. 利用权利要求1-8中任一所述方法制备获得的NRF2基因增强型的多能干细胞。
  11. 权利要求9所述的NRF2基因增强型的间充质干细胞或权利要求10所述的NRF2基因增强型的多能干细胞在如下c1)-c5)中任一种中的应用：
    c1)抵抗和/或延缓细胞衰老；
    c2)抵抗肿瘤发生；
    c3)抵抗恶性成瘤转化；
    c4)细胞移植治疗；
    c5)再生和/或修复损伤血管。
  12. 权利要求9所述的NRF2基因增强型的间充质干细胞或权利要求10所述的NRF2基因增强型的多能干细胞在如下d1)-d5)中任一种中的应用：
    d1)制备抵抗和/或延缓细胞衰老的产品；
    d2)制备抵抗肿瘤发生的产品；
    d3)制备抵抗恶性成瘤转化的产品；
    d4)制备细胞移植治疗的产品；
    d5)制备再生和/或修复损伤血管的产品。
  13. 一种产品，其活性成分为权利要求9所述的NRF2基因增强型的间充质干细胞；
    所述产品的功能为如下m1)-m5)中任一种：
    m1)抵抗和/或延缓细胞衰老；
    m2)抵抗肿瘤发生；
    m3)抵抗恶性成瘤转化；
    m4)细胞移植治疗；
    m5)损伤血管的再生和/或修复。
  14. 根据权利要求11或12所述的应用或权利要求13所述的产品，其特征在于：
    所述抵抗和/或延缓细胞衰老体现在如下n1)-n6)中任一种：
    n1)在外源刺激物刺激下细胞活力增强；
    n2)在体外连续传代培养过程中表现更强的增殖能力；
    n3)清除有害代谢物的能力增强；
    n4)与细胞衰老相关基因的表达水平降低；
    n5)具有更完整的细胞核结构；
    n6)在人或动物体内存留时间更长。
  15. 根据权利要求11或12所述的应用或权利要求13所述的产品，其特征在于：
    所述抵抗恶性成瘤转化体现在如下e1)-e3)中的任一种：
    e1)瘤化细胞增殖能力降低；
    e2)与肿瘤形成相关的非锚定依赖的生长能力降低；
    e3)在体内形成肿瘤的能力降低。
  16. 根据权利要求11或12所述的应用或权利要求13所述的产品，其特征在于：所述细胞移植治疗为细胞移植修复治疗。
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