CN109762845B - Mesenchymal stem cell model with function loss of RAP1 and construction method and application thereof - Google Patents
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Abstract
The invention discloses a mesenchymal stem cell model with a lost RAP1 function, a construction method and application thereof. The preparation method of the mesenchymal stem cell model with the functional loss of RAP1 provided by the invention comprises the following steps: reducing the content and/or activity of RAP1 in the pluripotent stem cell or inhibiting the expression of RAP1 encoding gene in the pluripotent stem cell to obtain the pluripotent stem cell with the function of RAP1 lost; inducing RAP1 dysfunction-free pluripotent stem cells to obtain RAP1 dysfunction-free mesenchymal stem cells; the pluripotent stem cells are embryonic stem cells or induced pluripotent stem cells. The mesenchymal stem cell model with the RAP1 loss function shows that the cell proliferation capacity is improved, the degenerative speed is reduced, the in-vivo retention capacity is improved, and the telomere length is prolonged, so that the mesenchymal stem cell model can be used for researching the relation between telomere length change and cell aging, screening toxic molecules for accelerating cell aging and serving as a cell material with enhanced proliferation and in-vivo retention capacity for cell treatment.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a RAP1 loss-of-function mesenchymal stem cell model, a preparation method and application thereof, and particularly relates to a RAP1 loss-of-function human embryonic stem cell model, a RAP1 loss-of-function human mesenchymal stem cell model, a preparation method and application thereof.
Background
Telomere DNA is a DNA repetitive sequence at the tail end of a linear chromosome of a eukaryote, forms a high-level structure together with a complex protein component recruited by the telomere DNA, maintains the stability of the tail end of the chromosome, and has important physiological significance. The abnormal telomere can cause the chromosome instability of cells, and cause DNA damage and repair reaction, thereby causing the abnormal recombination of the chromosome, terminal adhesion and the like. With DNA replication and cell division, the "theory of senescence" states that over-shortening of telomeres will lead to cell viability decline and loss of division ability, leading to replicative senescence of cells and, in turn, to senescence in the body.
The adult stem cells are a general term of cells existing in an organism, have self-renewal and certain differentiation potential, play an important role in maintaining the steady state of the environment in the organism and supplementing lost multiple terminal differentiated cells, and are closely related to the aging of the organism in terms of the imbalance, decline and exhaustion of the steady state of the adult stem cells. Mesenchymal Stem Cells (MSCs) are important members of adult stem cells, are originally derived from early-developing mesoderm and ectoderm, have self-replicating ability and ability to differentiate into various cells such as fat, muscle, cardiac muscle, cartilage, osteogenesis, and the like, and are also important for the regulation of inflammatory responses in the body, and abnormal or accelerated depletion of Mesenchymal stem cells is an important cause of osteoporosis, muscle atrophy, and some premature aging. In the application aspect, a system based on separation, in vitro differentiation and human mesenchymal stem cell culture is mature, and the mesenchymal stem cells have the advantages of important functions, functions and the like, and become important tool cell types in the field of cell therapy. Research shows that the mesenchymal stem cells cultured in vitro obviously show a replicative senescence phenotype. In conclusion, artificial regulation of telomere length in human mesenchymal stem cells is of great importance: (1) in the aspect of scientific research, telomeres and mesenchymal stem cells are closely related to replicative senescence, senescence is always an interesting topic for human beings, population aging is a serious problem facing the world, particularly China, and research on the telomeres by taking the mesenchymal stem cells as a model can help people to further understand senescence and realize assistance to 'healthy senescence'. Meanwhile, because the telomere length and the regulation mechanism of model organisms such as nematodes, mice and the like are obviously different from those of human beings, and the telomere of tumor cells is obviously different from that of normal cells, the research in the human mesenchymal stem cells is helpful for people to better understand the action and the regulation mechanism of the telomere in the human physiological state. (2) In the application aspect, the mesenchymal stem cells with higher activity and in-vivo retention capacity are obtained through gene editing, and can be used for assisting in cell therapy by using the mesenchymal stem cells. For mesenchymal stem cells with a replicative senescence phenotype, lengthening telomeres may be one of the important means to enhance their viability. At present, the simplest method for lengthening telomeres is to over-express telomerase, but the potential risk of canceration exists due to over-activation of the telomerase, so that the search for other safer methods for regulating the length of the telomeres becomes an important problem.
The RAP1 (prepromator protein 1) gene, also known as TERF2IP (TERF2interacting protein), is located on human chromosome 16q23.1 for a total of 3 exons. The RAP1 protein is approximately 50kDa and contains 399 amino acids.
Disclosure of Invention
The invention aims to solve the technical problem of how to prepare a cell model with prolonged telomere, enhanced in-vivo retention capacity and proliferation capacity and delayed aging process.
In order to solve the above technical problems, the present invention first provides a method for preparing a cell model, the method comprising the steps of: reducing the content and/or activity of RAP1 in the stem cells or inhibiting the expression of RAP1 encoding genes in the stem cells to obtain a cell model.
In the above method, the stem cell may be a mesenchymal stem cell or a pluripotent stem cell.
When the stem cell is a pluripotent stem cell, the method for preparing the cell model may comprise the steps of: reducing the content and/or activity of RAP1 in the pluripotent stem cell or inhibiting the expression of RAP1 encoding gene in the pluripotent stem cell to obtain the pluripotent stem cell with the function of RAP1 lost; and (3) inducing the RAP1 pluripotent stem cell with the loss of function to obtain a mesenchymal stem cell model.
Further, the method for inducing said pluripotent stem cells with loss of function of RAP1 may comprise the steps of: and (3) performing embryoid body differentiation on the RAP1 dysfunction-lost pluripotent stem cell, and screening cells which are all positive for CD73, CD90 and CD105 to obtain the cell model.
Further, the method for inducing said pluripotent stem cells with loss of function of RAP1 can be specifically performed according to the following steps: performing embryoid body differentiation (differentiation time can be 48-72 hours) on the RAP1 function-lost pluripotent stem cell to obtain an Embryoid Body (EB); the EBs were then plated on Matrigel (Matrigel) -coated plates and cultured until fibroblasts appeared (culture time could be 2 weeks). And after one passage, sorting cell populations in which the CD73, the CD90 and the CD105 are all positive by using flow cytometry to obtain the cell model.
In the above method, the pluripotent stem cell may be an embryonic stem cell or an induced pluripotent stem cell.
Further, the embryonic stem cell or induced pluripotent stem cell may be a human embryonic stem cell or a human induced pluripotent stem cell.
Further, the human embryonic stem cell may be a commercial human embryonic stem cell, such as human embryonic stem cell H9.
In the above method, the reduction of the content and/or activity of RAP1 in the stem cell can be performed by any one of the following methods: inhibiting synthesis of RAP1 protein in stem cells, inhibiting function of RAP1 protein in stem cells, and promoting degradation of RAP1 protein in stem cells. The inhibition of expression of the gene encoding RAP1 in the stem cell can be achieved by any one of the following means: RNA interference, gene silencing, gene knockout, gene mutation.
Further, the reduction of the content and/or activity of RAP1 in the stem cell can be realized by adding an RAP1 protein inhibitor into the stem cell. The RAP1 protein inhibitor can be a protein, polypeptide or small molecule compound that inhibits synthesis of RAP1 protein or inhibits the function of RAP1 protein or promotes degradation of RAP1 protein.
The inhibition of the expression of the RAP 1-encoding gene in the stem cell can be realized by adding RNA which can interfere with the expression of the RAP 1-encoding gene into the stem cell, and can also be realized by adding a substance for knocking out or mutating the RAP 1-encoding gene into the stem cell.
Further, said inhibiting the expression of the gene encoding RAP1 in the stem cell is achieved by knocking out the gene encoding RAP1 in the stem cell. The RAP1 encoding gene in the knockout stem cell can be realized by a CRISPR/Cas9 method. The gene encoding RAP1 in the knockout stem cell may specifically be exon 2 of the gene encoding RAP1 in the knockout stem cell.
In one embodiment of the present invention, the method for knocking out exon 2 of the RAP1 encoding gene using the CRISPR/Cas9 method may comprise the steps of: and (3) introducing a gRNA targeting the No. 2 exon, Cas9 and a DNA fragment (marked as DNA fragment 1) containing the No. 2 exon upstream and downstream homology arms into the stem cell to knock out the No. 2 exon of the RAP1 encoding gene.
The target sequence of the gRNA can be specifically a sequence 3 in a sequence table.
The sequences of the upstream and downstream homology arms can be specifically a sequence 1 and a sequence 2in a sequence table.
The DNA fragment 1 also contains a neo resistance gene. The DNA fragment 1 sequentially comprises a left arm of a homologous arm shown in 21 st-1461 st position of a sequence 1 in a sequence table, a neo resistance gene and a right arm of a homologous arm shown in 21 st-1259 th position of a sequence 2in the sequence table.
Knocking out exon 2 of the RAP1 encoding gene using the CRISPR/Cas9 method may further comprise the steps of: and introducing a vector for expressing the gRNA, a vector for expressing Cas9 and a vector containing the DNA fragment 1 into the stem cell to knock out the No. 2 exon of the RAP1 encoding gene.
The vector for expressing the gRNA can be a recombinant vector obtained by integrating a DNA fragment shown in a sequence 4 in a sequence table into a gRNA cloning vector by a homologous recombination connection method. The recombinant vector can express a gRNA capable of targeting exon 2 of the RAP1 encoding gene.
The vector containing the DNA fragment 1 can be a recombinant vector obtained by replacing a DNA fragment between Apa I and Xho I recognition sequences of pCR2.1-neo with a DNA fragment shown in 21-1461 bit of a sequence 1 in a sequence table, and replacing a DNA fragment between SacI and Kpn I recognition sequences of pCR2.1-neo with a DNA fragment shown in 21-1259 bit of a sequence 2in the sequence table, wherein the recombinant vector contains the left arm of the homology arm shown in 21-1461 bit of the sequence 1 in the sequence table and the right arm of the homology arm shown in 21-1259 bit of the sequence 2in the sequence table, and a neo resistance gene is also contained between the left and right homology arms.
After the vector for expressing the gRNA, the vector for expressing Cas9 and the vector containing the DNA fragment 1 are introduced into the stem cell, the method further comprises the steps of screening and identifying positive clones, and specifically comprises the following steps:
1) adding G418 into a cell culture system, and screening to obtain neo-positive clones;
2) after the step 1) is finished, primarily screening the human embryonic stem cell clone successfully edited at the genome level by adopting a PCR technology, and carrying out amplification culture on the human embryonic stem cell clone;
3) after the step 2) is finished, carrying out electric transformation by using a vector capable of expressing FLPo recombinase, and screening positive clones by using puro;
4) and 3) after the step 3) is finished, selecting positive clone amplification, and carrying out identification and verification by using a PCR technology again to obtain a clone with a correct identification result.
The invention also provides any one of the following X1) -X6):
x1) a cell model obtained by the method for producing a cell model; the cell model has the phenotypes of prolonged telomere length, improved cell proliferation capacity, improved in-vivo retention capacity and delayed aging process;
x2) a cell model for screening for substances which influence the telomere length and/or the cell proliferation capacity and/or the in vivo persistence capacity and/or the progression of cellular senescence of cells, being said cell model;
x3) a system for constructing the cell model consisting of a gRNA targeting the RAP1 encoding gene and a Cas9 nuclease;
x4) a system for constructing the cell model, consisting of a vector expressing X3) the gRNA and a vector expressing Cas9 nuclease;
x5) a system for constructing the cell model consisting of a gRNA targeting the 2 nd exon, Cas9 nuclease and a DNA fragment containing the 2 nd exon upstream and downstream homology arms (the DNA fragment 1);
x6) for constructing the cell model, consisting of a vector expressing the gRNA, a vector expressing Cas9 nuclease, and a vector containing a DNA fragment of the 2 nd exon upstream and downstream homology arms (the DNA fragment 1).
X3) and X4) can specifically target exon 2 of the gene encoding RAP 1.
The invention also provides the application of the cell model in any one of the following Y1) -Y6):
y1) as a cell model with enhanced proliferation and/or in vivo retention capacity;
y2) in a cell model as telomere elongation;
y3) as a cell model for the delay of aging process;
y4) for screening for substances which influence the telomere length and/or the cell proliferation capacity and/or the in vivo persistence capacity and/or the progression of cell senescence;
y5) for screening for substances that shorten telomere length and/or inhibit cell proliferation and/or inhibit cell persistence in vivo and/or accelerate cell senescence;
y6) for the preparation of a product for cell therapy.
In the above application, the cell may be a stem cell. The stem cells may specifically be adult stem cells.
In the above application, the substance may be a drug and/or a natural organic substance and/or a small molecule compound and/or a toxic molecule.
In the above application, the product for cell therapy may specifically be a cell therapy product with extended telomere length or a cell therapy product with enhanced proliferative capacity or a cell therapy product with delayed senescence process or a cell therapy product with enhanced in vivo retention capacity.
In the above method or product or application, the sequence of the RAP1 encoding gene may be GenBank: positions 75,647,737-75,657,442 of NC-000016.10 (the genomic sequence of the RAP1 gene), updated on PRI 12-JUL-2017; the sequence of the RAP1 encoding gene can also be GenBank: NM-018975.3 (cDNA sequence of RAP1 gene), updated on PRI 10-JUL-2017.
Experiments prove that the cell model of the invention has the advantages of prolonged telomere length, improved cell proliferation capacity, improved in vivo retention capacity and delayed aging process. Therefore, the cell model of the invention can be used for researching the relation between telomere length change and cell aging, screening toxic molecules which enable cells to accelerate aging and being used for cell therapy as cell materials with enhanced proliferation and in-vivo retention capacity.
Drawings
FIG. 1 shows that human embryonic stem cells (hESCs) of the invention, which have lost the function of RAP1, are RAP1-/-hESC. A is the gene editing strategy diagram of the 2 nd exon of RAP1 in human embryonic stem cells; b is a genome PCR detection result of RAP1 related targeting identification; c is immunofluorescence detection RAP1-/-Stem cell characteristics of hescs; d is Western blotting detection wild type and RAP1-/-RAP1 protein expression levels in hescs. RAP1-/-Representing RAP1-/-hESC，RAP1+/+H9 cell line.
FIG. 2 is a RAP1 related to the present invention-/-hESC-derived RAP1-/-hmscs have a phenotype of enhanced proliferative and in vivo retention. Wherein A is RAP1+/+hMSC and RAP1-/-Sorting results of flow cytometry of hMSC positive surface marker proteins CD105, CD73 and CD 90; b is identification of RAP1 by Western blotting+/+hMSC and RAP1-/-Expression level of RAP1 protein in hMSC, RAP1-/-Loss of RAP1 expression in hmscs; c is RAP1+/+hMSC and RAP1-/-Proliferation profile of hMSC cells; d is the senescence-associated β -Gal staining result; e is RAP1-/-Testing the retention capability of hMSC in vivo. RAP1-/-Representing RAP1-/-hMSC，RAP1+/+Wild-type hmscs are indicated.
FIG. 3 is a RAP1 related to the present invention-/-hmscs have a telomere-extended phenotype. A, detecting the length of a telomere restriction fragment by using a Southern blotting method; b for telomere length detection using real-time quantitative PCR, RAP1-/-hmscs are longer in telomere length. RAP1-/-Representing RAP1-/-hMSC，RAP1+/+Wild-type hmscs are indicated.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents, instruments and the like used in the following examples are commercially available unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.
The cell culture conditions in the following examples were all 37 ℃ and 5% CO2。
The following examples of fluorescently labeled antibodies used for flow cytometry sorting of hmscs are as follows:
fluorescein FITC labeled anti-human cell surface recognition molecule CD90 antibody (555595), BD Biosciences.
Fluorescein PE-labeled anti-human cell surface recognition molecule CD73 antibody (550257), BD Biosciences.
Fluorescein APC labeled anti-human cell surface recognition molecule CD105 antibody (17-1057-42), eBiosciences.
Fluorescein APC was labeled with isotype control antibody (555751), BD Biosciences.
Fluorescein PE labeled isotype control antibody (555749), BD Biosciences.
Fluorescein FITC labeled isotype control antibody (555742), BD Biosciences.
The human embryonic stem cell H9 cell line (product of WiCell corporation, cat. No.: WA09(H9) -DL-7) in the examples described below.
The cell culture media formulations in the following examples were as follows:
(1) CDF12 medium formula:
DMEM/F12 medium (Invitrogen, cat # 11320-033);
0.1mM non-essential amino acid (Invitrogen, Cat: 11140-050);
1mM GlutaMAXTMdipeptide (Invitrogen, Cat. No.: 35050-;
20% (by volume) Knockout serum replacement (Invitrogen, cat # N10828-028);
1% (1g/100ml) penicillin/streptomycin (Invitrogen, cat. No.: 15070-;
55 μ M of β -mercaptoethanol (Invitrogen, Cat. No. 21985-023);
10ng/ml of human FGF2 (product of Joint Protein Central Co.).
(2) Mesenchymal Stem Cell (MSC) medium formulation:
MEM medium (product of Invitrogen corporation, cat # 12571071);
10% (volume percentage content) fetal bovine serum (product of Gemcell company, cat # 100-;
0.1mM non-essential amino acid (Invitrogen, Cat: 11140-050);
1mM GlutaMAXTMdipeptide (Invitrogen, Cat. No.: 35050-;
1% (1g/100ml) penicillin/streptomycin (Invitrogen, cat. No.: 15070-;
10ng/ml of human FGF2 (product of Joint Protein Central Co.).
The viral vectors expressing Luciferase in the following examples are described in the literature "Pan, h., Guan, d., Liu, x., Li, j., Wang, l., Wu, j., Zhou, j., Zhang, w., Ren, r., Li, y., et al. (2016.," SIRT6 secure human genetic stem cells from oxidative stress by coactive mutation nrf2. research 26,190-205 ", from which the applicant publicly available the biomaterial only for repeating the relevant experiments of the present invention, and not for other uses.
The pCR2.1-neo vector in the examples given below is given by professor Juan Carlos IZPisua Belmonte, national institute of Salk, Japan, Yuan, G, Liu, X, Ren, R, Li, J, Zhang, W, Wu, J, Xu, X, Fu, L, Li, Y, et al (2015) PTEN default reproduction hum human neural cells, devices of which the contents are listed in the publications "Duan, S, Yuan, G, Liu, Y, et al", "U, X, Fu, L, Li, Y, et al", "com communications 6,10068", and "Pan, H, Guan, D, Liu, X, Li, J, Wang, L, Wu, J, Zhang, W, Z, W.S. biological materials of which the applicant is a biological research institute of the family of the species of the present invention, and is repeatedly available from the inventor, biological materials of the family, research, Inc, research, the family, research, the inventor, biological materials of the family, Wei, research, Hu, III, research, it is not usable for other purposes.
The Cas9 expression vectors in the following examples were given by professor George m.church, described in the literature "Mali, p., Yang, l., envlt, k.m., Aach, j., Guell, m., DiCarlo, j.e., Norville, j.e., and Church, G.M. (2013). RNA-guided human genome engineering via case 9.science 339, 823-826", and were maintained by the first inventor at the institute of biophysics of the chinese academy of sciences, and were publicly available from the applicant only for the repetition of experiments related to the present invention and not for other uses.
The FLPo recombinase expression vectors in the following examples are described in the literature "Liu, g.h., Qu, j., Suzuki, k., Nivet, e., Li, m., Montserrat, n., Yi, f., Xu, x, Ruiz, s., Zhang, w., et al (2012), Progressive production of human neural stem cells used by pathogenic lrrk2. natural 491, 603-607", and are kept by the first inventor at the institute of biophysical research of the chinese academy of sciences, from which the public can obtain the biological material only for use in repeating experiments related to the present invention, and cannot be used by the applicant as other uses.
Example 1 construction of RAP1 loss-of-function embryonic stem cell line and identification thereof
This example relates to the targeted inactivation of human RAP1 gene (genome sequence: GenBank: 75,647,737-75,657,442 of NC-000016.10, Updated on PRI 12-JUL-2017; cDNA sequence: GenBank: NM-018975.3, Updated on PRI 10-JUL-2017) in human embryonic stem cells to obtain human embryonic stem cells with loss of RAP1 protein function and RAP1 function.
The invention firstly designs and obtains homologous arm sequences at two sides of a 2 nd exon of RAP1 gene in a targeted human genome by a molecular cloning method, and then constructs the homologous arm sequences on a pCR2.1-neo vector to obtain a RAP1 homologous arm vector. Then, a gRNA Vector targeting the 2 nd exon of the RAP1 gene was constructed (Addgene, product name and product number are gRNA Cloning Vector, Plasmid #41824, respectively). And finally, electrically transforming the RAP1 homologous arm vector, the gRNA expression vector and the Cas9 expression vector into a human embryonic stem cell together to obtain the RAP1 human embryonic stem cell with the 2 nd exon deleted (figure 1A). Wherein, after the human embryonic stem cells are electrically transformed, the method also comprises the step of cloning and screening by using G418; after the clone screening, the method also comprises a step of confirming the homologous recombination condition of the selected clone by using a method of genome PCR. The specific method comprises the following steps:
first, construction of RAP1 loss-of-function embryonic stem cell line
1. Construction of gRNA expression vector
A gRNA coding gene targeting RAP1 gene is connected into a gRNA cloning vector of Addgene company to construct a gRNA expression vector for knocking out RAP1 gene. The method comprises the following specific steps:
1) gRNA sequences targeting exon 2 of the RAP1 gene were designed according to the RAP1 genomic data search analysis provided by NCBI. The target sequences were designed as follows: TGGGTGAATGAGCACGTCCT-AGG (SEQ ID NO: 3).
2) Human genome DNA is taken as a template, RAP1gRNA-F and RAP1gRNA-R primers are adopted for PCR amplification, and a gRNA sequence is obtained. The primer sequences are as follows (the sequences shown underlined are the target sequences):
RAP1gRNA-F：
TTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCTGGGTGAATGAGCACGTCCT；
RAP1gRNA-R：
GACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAACAGGACGTGCTCATTCACCCA。
the PCR amplification system is as follows: 5 XGC buffer 10. mu.L, 2.5mM dNTP 4. mu. L, RAP1gRNA-F (10. mu.M) 2. mu. L, RAP1gRNA-R (10. mu.M) 2. mu. L, Phusion polymerase (product of NEB Co., cat. No.: M0530L) 0.5. mu.L, ddH2O 31.5μL。
The PCR reaction conditions were as follows:
after completion of the reaction, the gRNA sequence was recovered using a PCR recovery kit (QIAquick Co., Ltd., cat # 28106).
3) The gRNA cloning vector is cut by Afl II enzyme, the recovered vector fragment is subjected to homologous recombination and connection with a gRNA sequence recovered by a PCR recovery kit, the obtained recombinant vector with a correct sequence is named as gRNA-RAP1, and the gRNA-RAP1 contains a DNA fragment shown in a sequence 4 in a sequence table.
The ligation reaction (20. mu.L) was as follows:
note: the values of x and y are determined according to the concentrations of the gRNA enzyme digestion vector and the gRNA PCR recovery product. The molar ratio of the carrier fragment and the gRNA sequence after the general gRNA enzyme digestion recovery is 1:3-1: 10. 2 × Gibson
Master Mix is a product of NEB corporation.
The ligation reaction conditions were as follows: 50 ℃ for 1 hour.
2. Construction of homology arm vectors
Designing left and right homologous arms according to the position of gRNA, and constructing the left and right homologous arms into pCR2.1-neo vectors, wherein the left and right homologous arms are respectively positioned at the upstream and downstream of the 2 nd exon of RAP1 gene in a human cell genome. The method comprises the following specific steps:
1) carrying out PCR amplification on human genome DNA by using RAP1Left Arm Primer F and RAP1Left Arm Primer R to obtain a Left Arm of a homologous Arm (the sequence is a sequence 1 in a sequence table); human genome DNA was PCR amplified using RAP1Right Arm Primer F and RAP1Right Arm Primer R to obtain the Right Arm of the homology Arm (sequence 2in the sequence Listing). The enzyme used for PCR amplification was PrimeSTAR (product of TaKaRa Co., Ltd.; cat. DR 010A). The primer sequences are as follows:
| RAP1Left Arm Primer F | ctatagggcgaattgggcccAGCCTCTATTACCGTCTCTTGTCTGTTGCAT |
| RAP1Left Arm Primer R | ctggcggccgctcgaGGGCCACGTACCACAATCCACCAATATACCAT |
| RAP1Right Arm Primer F | ttactagtggatccgagctcTGGAAAATGGGACTGATCTGGGCTTCAGAC |
| RAP1Right Arm Primer R | attacgccaagcttggtaccTCACCACATCTCCAATACCCACCAATGCCTA |
2) the pCR2.1-neo is cut by Apa I and Xho I to obtain a pCR2.1-neo vector skeleton; the DNA fragment containing the left arm of the homology arm was ligated to pCR2.1-neo vector backbone using PCR one-step directed cloning kit (NovoRec: NR001), and the resulting recombinant vector with the correct sequence was named pCR2.1-neo-RAP 1-u.
Carrying out enzyme digestion on pCR2.1-neo-RAP1-u by using SacI and KpnI to obtain a pCR2.1-neo-RAP1-u vector skeleton; the DNA fragment containing the right arm of the homology arm was ligated to the vector backbone pCR2.1-neo-RAP1-u, and the resulting recombinant vector with the correct sequence was named pCR2.1-neo-RAP 1.
pCR2.1-neo-RAP1 contains a DNA fragment (left arm of homology arm) shown at 21 st-1461 st position of sequence 1 in the sequence table and a DNA fragment (right arm of homology arm) shown at 21 st-1259 st position of sequence 2in the sequence table, and a neo resistance gene is also contained between the left and right homology arms.
3. Acquisition of human embryonic stem cells with loss of RAP1 function
CRISPR/Cas 9-based gene targeting technology is used for specifically knocking out exon 2 of RAP1 gene in human embryonic stem cells. The method comprises the following specific steps:
1) culturing a human embryonic stem cell H9 cell line by a method comprising the following steps:
a. the human embryonic stem cell H9 cell line was inoculated into a culture plate in which mouse embryonic fibroblasts (Invitrogen, product: S1520-100) inactivated by mitomycin (Selleck, product: S8146) were previously cultured, and cultured together with the mouse embryonic fibroblasts using a human embryonic stem cell medium (CDF12 medium);
b. the human embryonic stem cell H9 cell line was seeded on a culture plate previously coated with an extracellular matrix (BD-Biosciences, cat # 354277) and cultured using mTeSR medium (StemShell Technologies).
2) H9 cells proliferating in log phase were washed once with PBS, digested with Tryple Express (Invitrogen corporation, cat # 12604021) for 5-10 minutes, and then gently blown into single cells.
3) Subjecting the 5X 10 of step 2) to6H9 unicell, configuring Cell suspension according to Cologne _ CoA _ cGMP Solution P3Primary Cell 4D-Nucleofector Kit (product of Lonza corporation, goods number: V4XPG-3024), adding Cas9 expression vector, gRNA-RAP1 vector obtained in step 1 and pCR2.1-neo-RAP1 vector obtained in step 2 into the Cell suspension by 7 μ g each, and using 4D electric converter (product of Lonza corporation, goods number: 4D-Nucleofector)TMSystem) to perform electrical transfer.
4) The electroporated cells were added to culture plates containing MEF. The fresh CDF12 medium was changed after 24 hours.
5) After about 2 days, after the clone grows out of the culture plate, G418 is added into a cell culture system according to the clone number and size, the neo positive clone is obtained through screening, the successfully edited human embryonic stem cell clone at the genome level is preliminarily screened by combining the PCR technology, the clone is enlarged and cultured, 10 mu G of carrier capable of expressing FLPo recombinase is used for electric transformation, puro screening is used, the positive clone is picked for amplification, the PCR technology is used again for identification and verification, and the clone with the correct identification result is obtained, namely the human embryonic stem cell with the RAP1 loss function (marked as RAP 1)-/-hESC). The primers used in the PCR technique are the same as the identification primers used in step 1.
II, identification of RAP 1-disabled human embryonic stem cells
Whether the generated clone is correctly targeted is identified by means of quantitative PCR, Western blotting and the like, and the method specifically detects the clones from the following aspects: and (3) detecting whether the 2 nd exon of the RAP1 gene is deleted and whether the RAP1 protein is deleted in the genome.
1. PCR identification
Extraction of wild type (H9 cell line) and RAP1-/-hESC genomic DNA, whether exon 2 was deleted was identified using PCR as the primer. The primer sequences are as follows:
P1：5’-TTGGCAAAAGTCAATACAATGGGTAATATCCAAAG-3’；
P2：5’-TTTGACTTCACTCTCAAGACTGTAAGCTCCT-3’；
P3：5’-GTGGATTGTGGTACGTGGCCCAGATCTGCC-3’；
P4：5’-TAACATACCACAACCTCCTCAAACTCCCGG-3’。
wherein P1+ P2 spans the left and right homology arms, and the length of the band obtained by PCR is shortened after the 2 nd exon of RAP1 is deleted. Among them, P4 is inside the 2 nd exon of RAP1, and after this exon is deleted, P3+ P4 could not obtain PCR product. The PCR identification result is shown as B in FIG. 1, and the result shows RAP1-/-Exon 2 of RAP1 has been deleted in hESC.
2. Immunofluorescence detection of stem cell characteristics of modified human embryonic stem cells
With H9 cell line and RAP1-/-The two types of hESC are used as test cells, and molecular markers OCT4, SOX2 and NANOG related to the dryness maintenance of human embryonic stem cells are detected by adopting an immunofluorescence technique. The method comprises the following specific steps:
the test cells cultured on the coverslip were fixed with 4% paraformaldehyde at room temperature for 30 minutes, rinsed with PBS (3 times, 5 minutes/time), and then incubated with PBS containing 0.4% (by volume) Triton X-100 at room temperature for 30 minutes, followed by blocking with 10% (by volume) donkey serum (Jackson ImmunoResearch Laboratories, Inc., cat # 017-000-one 121) at room temperature for 1 hour. After which the cells were incubated overnight at 4 ℃ with blocking solution supplemented with primary antibody. After PBS rinsing (3 times, 5 min/time), the corresponding secondary antibody was then added and incubated for 1 hour at room temperature. After PBS rinsing (3 times, 5 minutes/time), incubation was carried out with Hoechst 33342 (product of Life technology, cat # H3570) at a working concentration of 2. mu.g/ml for 15 minutes at room temperature, and finally mounting and observation were carried out.
The results are shown in FIG. 1, C, similar to the H9 cell line,RAP1-/-hESC are capable of expressing three molecular markers OCT4 (green fluorescence), SOX2 (red fluorescence) and NANOG (yellow fluorescence).
The above results indicate that loss of RAP1 function has no significant effect on the expression of stem cell sternness genes.
3. Western blotting identification of RAP1 protein
With H9 cell line and RAP1-/-The two types of hESC cells were used as test cells, and total proteins of the cells were extracted and proteins expressed by the cells were detected by Western blotting. The primary antibody used was RAP1 antibody (anti-RAP1, murine monoclonal, product of Santa Cruz Co., Ltd.; cat # sc-53434), and the secondary antibody was HRP-labeled goat-anti-mouse antibody (product of Santa Cruz Co., Ltd.; cat # sc-2005). Beta-actin is used as an internal reference, a primary antibody is a mouse-derived anti-beta-actin antibody (product of Santa cruz company, cat # sc-8432), and a secondary antibody is an HRP-labeled goat anti-mouse antibody (product of Santa cruz company, cat # sc-2005).
The result of detecting RAP1 protein by Western blotting is shown in D in FIG. 1, RAP1-/-hESC cells failed to detect expression of RAP1 protein with RAP1 antibody; and the H9 cell line can detect the expression of RAP1 protein (molecular weight is about 50kDa) by using RAP1 antibody.
Example 2, RAP1-/-hESC in vitro directed differentiation for RAP1-/-hMSC and RAP1-/-Phenotypic characterization of hMSCs
This example will use the RAP1 obtained in example 1-/-Further directed differentiation of hESCs into mesenchymal stem cells in vitro (RAP 1)-/-hMSC) and found RAP1-/-hmscs are able to delay the senescence phenotype to some extent.
First, RAP1-/-hESC in vitro directed differentiation for RAP1-/-hMSC
Mix RAP1-/-hESC undergoes Embryoid Body (EB) differentiation for 48-72 hours to obtain Embryoid Body (EB). The EBs were then seeded in 6-well plates coated with Matrigel (Invitrogen) and cultured for 2 weeks until the appearance of fibroblasts. After one passage, the cell groups with positive CD73, CD90 and CD105 (A in figure 2) are sorted by flow cytometry, namely the RAP1 functionLoss (RAP 1)-/-) Human mesenchymal stem cells (designated as RAP 1)-/-hMSC). Simultaneously, the H9 cell line is directionally induced and differentiated into the mesenchymal stem cells (marked as RAP 1)+/+hMSC)。
II, RAP1-/-Phenotypic characterization of hMSCs
1. Western blotting detection of abundance of RAP1 protein in MSC obtained by directional differentiation
With RAP1+/+hMSC and RAP1-/-Two kinds of hmscs were used as test cells, and Western blotting was performed to determine whether or not RAP1 protein was expressed in the cells according to the method in step two, step 3, example 1.
The result is shown in FIG. 2, B, RAP1-/-hMSC cells failed to detect expression of RAP1 protein with RAP1 antibody; and RAP1+/+hMSC cells detected the expression of RAP1 protein (molecular weight approximately 50kDa) using RAP1 antibody.
2. Determination of proliferation potency
RAP1 for serial passages using cell count statistics-/-hMSC cells and RAP1+/+The growth ability of hMSC cells, the results show, and RAP1-/-hMSC cell comparison, RAP1+/+hmscs showed a significant decrease in proliferative capacity (C in fig. 2). The specific operation steps are as follows:
1) statistical Serial passage of RAP1 Using cell counts-/-hMSC and RAP1+/+Cumulative cell proliferation fold of hmscs;
2) calculating the proliferation multiple of each generation of cells, namely the number of the cells at the end of each generation of culture/the number of the cells at the initial culture of each generation;
3) cumulative cell proliferation fold log2(P1 fold cell proliferation) + log2(P2 fold cell proliferation) + … + log2(P17 fold proliferation of cells).
The statistical results are shown in fig. 2, C, and the results show that: and RAP1+/+hMSC cell comparison, RAP1-/-The hMSC has stronger proliferation capacity, and is particularly shown as RAP1+/+When the proliferation rate of hMSC is slow, RAP1-/-hMSC can still maintain faster proliferation rate for several generations, and the generation of growth retardation is reached later than RAP1+/+hMSC。
3. SA-beta-Gal staining
Cell senescence-associated β -galactosidase staining is a method for staining senescent cells or tissues based on the upregulation of the level of SA- β -Gal (senescent-associated beta-galactosidase) activity during senescence.
Respectively with RAP1-/-hMSC cells and RAP1+/+hMSC cells were test cells (two passage P2 and P9 were set as experimental groups, respectively), and SA- β -Gal staining was performed:
1) seeding cells in 6-well plates at appropriate density;
2) when the cell density reaches 60-80%, washing the cells twice by PBS;
3) fixing with 2% paraformaldehyde and 0.2% isovaleraldehyde for no more than 5 min;
4) washing with PBS for 2 times;
5) staining solution was added and incubated overnight at 37 ℃ in the dark. The dyeing liquid has the following formula:
6) washing with PBS for 2 times;
7) hoechst 33342 (product of Life technology, cat #: h3570) Incubating for 5 minutes at room temperature in dark;
8) washing with PBS once;
9) and (4) observing under a microscope.
With X-Gal as a substrate, a dark blue product is generated under the catalysis of aging-specific beta-galactosidase. The senescence of the cells or tissues can be observed under a common light microscope, and the SA-beta-Gal staining positive cell ratio in the two groups of cells is further subjected to quantitative statistical analysis.
The results are shown in FIG. 2, D, for RAP1 generation 2 (Passage 2, P2)+/+hMSC and RAP1-/-The proportion of SA-beta-Gal staining positive cells in the hMSC has no obvious difference; while9 th generation (Passage 9, P9) RAP1-/-hMSC and RAP1+/+Compared with hMSC, the proportion of SA-beta-Gal staining positive cells is obviously less (P)<0.001). It can be seen that RAP1 appears upon cell passage-/-Senescence progression of hMSC compared to RAP1+/+hmscs were significantly delayed.
4、RAP1-/-Measurement of in vivo retention Capacity of hMSC cell mice
To verify the wild type (RAP 1)+/+hMSC) and RAP1-/-In vivo viability of hMSCs, RAP1 was first infected with a Luciferase-expressing viral vector, respectively+/+hMSC and RAP1-/-And (3) after infecting the hMSC cells for 3-5 days, digesting the two cells into single cells respectively, and then injecting the single cells into the left and right tibialis anterior muscles of an immunodeficient NOD-SCID mouse (a product of Beijing Wintolite laboratory animal technology Co., Ltd.), wherein the injection amount of the two cells is the same. The in vivo retention capacity of hmscs was reflected by measuring luciferase activity in the mouse left and right tibialis anterior muscles 2, 4, and 6 days after injection using a small animal in vivo imaging system (PE). The specific operation method comprises the following steps:
1) selecting cells with good growth state and cell density of 60-80%, and infecting a virus vector expressing Luciferase;
2) 3-5 days after infection, when the cells grow full, recording as day 0, and digesting the TrypLExpress into single cells;
3) cell count, every 1X 106The individual cells were resuspended in 100. mu.l PBS;
4) the equivalent amount of cells was taken, mixed with Luciferase substrate (D-Luciferase recovery, lotus salt, product of GOLDBIO Co.), and the relationship between the fluorescence intensity and the number of cells was measured using a microplate reader.
5) Injecting 100 mul of cell suspension into the tibialis anterior muscle of the mouse;
6) the mouse status was observed daily;
7) 2, 4, 6 days after transplantation, the mice were removed, intraperitoneally injected with Luciferase substrate, and analyzed using a small animal in vivo imaging system after anesthesia. The fluorescence intensity of 5 biological replicates was counted.
The results are shownRAP1-/-The luciferase activity of hMSC (right leg) in tibialis anterior muscle was higher than that of RAP1+/+hMSC (left leg) was significantly elevated in the tibialis anterior (E in FIG. 2), suggesting fusion with RAP1+/+hMSC compared to RAP1-/-hMSC has a slow in vivo degenerative rate and a strong retention ability.
Example 3, RAP1-/-Detection of hMSC telomere length
1. Analysis of telomere restriction fragment Length Using Southern blotting
To investigate whether loss of RAP1 function affects changes in telomere length, RAP1 was used+/+hMSC (wild type) and RAP1-/-hMSC cells (two generations of P2 and P9) were used as test cells, and the cells were collected by normal culture and digestion, and the whole genomic DNA was extracted and analyzed for the relative length of telomere restriction fragments using Southern blotting. The method comprises the following specific steps:
1) digesting and collecting cells, and centrifuging for 5 minutes at 1000 rpm;
2) washed once with PBS, and then, using a salting-out DNA extraction kit (product of mayo corporation, cat #: d3313-01) extracting the genome DNA of the tested cell to obtain a large amount of complete genome DNA;
3) hinf I (product of NEB, cat #: r0155) and Rsa i (product of NEB corporation, cat No.: r0167) overnight enzyme digestion of genomic DNA;
4) mu.g of the digested genomic DNA was sampled, subjected to electrophoresis using 0.8% agarose gel, and subjected to electrophoresis at 2V/cm for 24 hours; denaturing the DNA with a denaturing buffer containing 0.5M NaOH;
5) transferring DNA from the gel to a positively charged nylon membrane by using a capillary transfer membrane method and utilizing siphoning;
6) DIG Easy Hyb Granules (product of Roche Co., Ltd., product number: 11796895001) prehybridization in a hybridization oven at 42 ℃, and adding denatured telomere-specific probes for hybridization overnight;
7) Anti-Digoxigenin-AP, Fab fragments (product of Roche Co., Cat. No.: 11093274910) hybridization at room temperature for 1 hour;
8) the CDP-Star, ready-to-use (product of Roche, cat #: 12041677001) and observing the relative length of the telomere restriction fragments.
Southern blotting showed RAP1-/-Significant retrogradation of the telomere restriction fragment blot of hMSC, indicating RAP1-/-hmscs were longer in telomere length (a in fig. 3).
2. Analysis of telomere signal intensity using real-time quantitative PCR
To further confirm the Southern blotting results, RAP1 was used+/+hMSC (wild type) and RAP1-/-The hMSC cell is a test cell, and the telomere length is detected by using another method, namely real-time quantitative PCR, and the specific steps are as follows:
1) digesting and collecting cells, and centrifuging for 5 minutes at 1000 rpm;
2) washed once with PBS, and then centrifuged using a centrifugal column-type genomic DNA extraction kit (product of tiangen corporation, cat No.: DP318-02) extracting the genomic DNA of the test cells;
3) the following primers were used with SYBR qPCR mix (products of eastern co., cat #: QPS-201C) for real-time quantitative PCR detection. The primer sequences are as follows:
| |
|
| Tel | |
| 2 | TCCCGACTATCCCTATCCCTATCCCTATCCCTATCCCTA |
| 36B4u | CAGCAAGTGGGAAGGTGTAATCC |
| 36B4d | CCCATTCTATCATCAACGGGTACAA |
wherein Tel 1 and Tel 2 detect the abundance of telomere sequences in the genome, and 36B4u and D detect the abundance of a single copy gene 36B4 site in the genome. After the experiment, the Tel results were normalized with 36B4 as an internal reference.
Real-time quantitative PCR revealed RAP1-/-The telomere signal of hMSC is stronger, which indicates RAP1-/-hmscs were longer in telomere length (B in fig. 3).
The above results indicate on the one hand that in hmscs, RAP1 is a negative regulator of telomere length; on the other hand, the fact that the telomere is increased after the RAP1 is lost is probably an important reason for the enhancement of cell proliferation capacity and the delay of aging process. In conclusion, mesenchymal stem cells with longer telomere length can be obtained by knocking out RAP1 and differentiating in vitro under the condition of not changing the activity of telomerase, and the cells have better cell viability compared with wild type.
<110> institute of biophysics of Chinese academy of sciences
<120> RAP1 function-loss mesenchymal stem cell model and construction method and application thereof
<160> 4
<170> PatentIn version 3.5
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ttaattattt atcttgaagt gtttgtgtgt ctgcacaaag cttttcctca ttcttttcta 180
attgattgca ctgtatttca ttatgtgaat gaacagtaat ttacttttta ttatggagaa 240
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accttcacct gattttagtt catggcattt tttaaatcta tacttttcat cccaacttcc 360
tggactgttt tgaagcaaat gccagacatc atatcatttt atctatttta gcatgtatct 420
ctcaaagata agcctctttt agttactatt taagaatttt attaaataca tacagtaaaa 480
ttaacttttt atggtgtaaa attatatgca ttttaaaaac tttatttttt attgtggtaa 540
aatgtatata ccgtaaaatt tacagttcag atagtttttt aagtgttctg ttcagtggca 600
ttattaagtg cattcacatt gttgggcaac tgtcaccacc atctgtatct agaacttact 660
cattttctaa aactgaaact ccatgccttt tattattttc tgcttctagg ggtcagaaac 720
atgcctatta aacactaatt ccccatttct ccctcctctt agcccctgga aacaaccatt 780
ccgccttctg tttctatgaa ttcaatattt ctagatacct catacaagta gaatcatgta 840
atatttgccc ttttgtgtct ggcttatttc actttacata atatcttcaa agttcatcca 900
tattgtagca tgtgtcagaa tttgattcct ttttaaaggt gaataatatt ccattgtatg 960
tatatactac attttgtttt tccattaatg tatcaattga catttggctt atttccatct 1020
tttgtttatt ttgaataatg ctgctgtgaa catgggtata actagtatct gtttgagacc 1080
ctgctttcca ttattttagg tatatgccta gaaatgcaat tgctggatca tatggtaatt 1140
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tgaggaactg ctgtactctt ttccagttat atgtgtttta acataggtaa atttttgtaa 1260
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aaaaattggg tagaataggg acagttgtat aaacagtata aagcatccca ctttgagtac 180
tttaatgtca atatattttt caaaaagttt atagtaattg ttttttctat ttttttgagc 240
cagagtctgg ctctgtcgcc caggctggag tgcagtggtg cgatcttggc tcactgcaag 300
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ctgtcaccca ggctggagtg cagtggtgcc atctcagctc actgtagcct ccacctccca 600
ggttccagca attcttctgc ctcagcctcc tgggtaactg ggattacagg catgtgccat 660
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Claims (6)
1. The application of a mesenchymal stem cell model in the following Y1) or Y2):
y1) for screening for substances which influence the telomere length and/or the cell proliferation capacity and/or the in vivo persistence capacity and/or the progression of cell senescence; the use is a non-disease diagnostic and therapeutic use;
y2) for screening for substances that shorten telomere length and/or inhibit cell proliferation capacity and/or inhibit persistence in vivo and/or accelerate the progression of cell senescence; the use is a non-disease diagnostic and therapeutic use;
the preparation method of the mesenchymal stem cell model comprises the following steps: reducing the content of RAP1 in the human embryonic stem cells to obtain human embryonic stem cells with the loss of RAP1 functions; inducing the RAP1 loss-of-function human embryonic stem cell to obtain a RAP1 loss-of-function human mesenchymal stem cell, namely the mesenchymal stem cell model;
the human embryonic stem cell is a commercialized human embryonic stem cell.
2. The use according to claim 1, wherein said human embryonic stem cell is human embryonic stem cell H9.
3. Use according to claim 1 or 2, characterized in that: the reduction of the RAP1 content in human embryonic stem cells is achieved by knocking out the gene encoding RAP1 in stem cells.
4. Use according to claim 3, characterized in that: the RAP1 encoding gene in the knockout stem cell is realized by a CRISPR/Cas9 method.
5. Use according to claim 3, characterized in that: the gene encoding RAP1 in the knockout stem cell is exon 2 of the gene encoding RAP1 in the knockout stem cell.
6. Use according to claim 4, characterized in that: the target sequence of the CRISPR/Cas9 is shown as a sequence 3 in a sequence table.
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