CN111450098A

CN111450098A - Application of lamivudine in delaying senescence and preparation of cell model for screening anti-senescence drugs

Info

Publication number: CN111450098A
Application number: CN202010310737.9A
Authority: CN
Inventors: 曲静; 刘光慧; 张维绮; 毕诗佳; 王思; 宋默识
Original assignee: Institute of Zoology of CAS
Current assignee: Institute of Zoology of CAS
Priority date: 2020-04-20
Filing date: 2020-04-20
Publication date: 2020-07-28

Abstract

The invention discloses application of lamivudine in delaying senescence and preparation of a cell model for screening anti-senescence drugs. The preparation method of the cell model comprises the following steps: reducing the content of the SIRT7 protein in the stem cell and/or reducing the activity of the SIRT7 protein in the stem cell and/or inhibiting the expression of the coding gene of the SIRT7 protein in the stem cell and/or editing the coding gene of the SIRT7 protein in the stem cell to obtain a cell model with SIRT7 function loss. The mesenchymal stem cell model with SIRT7 function loss can be used for screening molecules which can lead cells to delay aging, and can be used for cell treatment as related materials with weakened proliferation and in-vivo retention capacity. The invention also screens out the micromolecule 3TC which can lead the cell to delay the senility, can be used for improving the phenotype of the cell model, such as the weakening of the cell proliferation capability, the reduction of the in vivo retention capability and the acceleration of the senescence process, has the function of delaying the senescence process and is used for treating related diseases.

Description

Application of lamivudine in delaying senescence and preparation of cell model for screening anti-senescence drugs

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a cell model for screening anti-aging drugs and a preparation method thereof.

Background

The Sirtuins family is a class of NAD + -dependent deacetylases that are highly conserved from archaea to mammals. The Sirtuins family plays an important role in different cellular processes, such as apoptosis, mitochondrial biosynthesis, lipid metabolism, and aging. SIRT7 is a Sirtuins member uniquely localized in the nucleolus and involved in processes such as ribosome biogenesis and stress response. SIRT7 regulates liver lipid metabolism, cardiac system homeostasis and adipogenesis. SIRT7 also acts as a transcription repressing factor regulating mitochondrial and cytoplasmic protein homeostasis. In recent years, SIRT7 has also been reported to be associated with genomic stability. For example, SIRT7 mediates deacetylation of the H3K18ac site and succinylation of the H3K122succ site, recruited to DNA double strand breaks, promoting chromatin remodeling and repair.

The SIRT7 gene, also known as Sirtuin 7(NAD-Dependent Protein Deacetylase Sirtuin-7), is located on chromosome 17q25.3, has a total genomic length of about 6.2kb, encodes 10 exons and 9 introns, and can be spliced into mRNA of about 1.7kb in length. The protein molecular mass of SIRT7 is about 44.9kDa, and the isoelectric point is about 9.8.

Human mesenchymal stem cells maintain other cell populations in the body and have the potential to differentiate into various cell lineages, such as chondrocytes, osteoblasts, adipocytes, and the like.

Lamivudine (3TC) is a reverse transcriptase inhibitor, which has been used in AIDS-related therapy.

Disclosure of Invention

The invention aims to provide application of lamivudine in delaying senescence and preparation of a cell model for screening anti-senescence medicaments.

The invention firstly protects the application of lamivudine in preparing medicaments; the function of the medicine is as follows (a) and/or (b) and/or (c) and/or (d) and/or (e) and/or (f) and/or (g) and/or (h):

(a) delaying aging;

(b) delaying cell aging;

(c) delaying the aging of the mesenchymal stem cells;

(d) delaying the senescence of the mesenchymal stem cells with SIRT7 loss of function;

(e) improving the clonogenic capacity of the SIRT7 function-lost mesenchymal stem cells;

(f) increasing the proliferative capacity of the SIRT7 function-lost mesenchymal stem cells;

(g) reducing the proportion of senescent cells in the mesenchymal stem cells with SIRT7 loss of function;

(h) reducing retrotransposon levels in SIRT7 loss-of-function mesenchymal stem cells.

The mesenchymal stem cell is a human mesenchymal stem cell.

The mesenchymal stem cell with SIRT7 loss function can be any one of the cell models (mesenchymal stem cell model) described in the following

The invention provides a preparation method of a cell model, which comprises the following steps: reducing the content of SIRT7 protein in the stem cell and/or reducing the activity of SIRT7 protein in the stem cell and/or inhibiting the expression of coding gene of SIRT7 protein in the stem cell and/or editing the coding gene of SIRT7 protein in the stem cell to obtain the recombinant cell, wherein the recombinant cell or the passage cell thereof is a cell model. The subculture cell may be specifically a cell obtained by subculturing the recombinant cell. The number of subcultures is 1 or more.

The invention provides a preparation method of a cell model, which comprises the following steps:

(1) reducing the content of the SIRT7 protein in the stem cell and/or reducing the activity of the SIRT7 protein in the stem cell and/or inhibiting the expression of the coding gene of the SIRT7 protein in the stem cell and/or editing the coding gene of the SIRT7 protein in the stem cell to obtain a recombinant cell;

(2) and taking the recombinant cell or the passage cell of the recombinant cell, and inducing and differentiating to obtain the cell or the passage cell thereof, namely the cell model.

The subculture cell of the recombinant cell may specifically be a cell obtained by subculturing the recombinant cell. The number of subcultures is 1 or more.

After induced differentiation, the resulting cells were designated as P1 generation cells. The passaged cell may be any one of the cells from generation P2 to generation P10, may be any one of the cells from generation P3 to generation P10, may be any one of the cells from generation P4 to generation P10, and may be more specifically the cells from generation P3, generation P4, generation P5 or generation P6.

(2) and taking the recombinant cell or the passage cell of the recombinant cell, and inducing and differentiating to obtain the mesenchymal stem cell or the passage cell thereof, namely the cell model.

The induced differentiation is induced differentiation into mesenchymal stem cells.

The mesenchymal stem cells are positive cells of CD73, CD90 and CD 105.

After induced differentiation, the resulting cells were used as P1-generation mesenchymal stem cells. The passaged cell may be any one of the cells from generation P2 to generation P10, may be any one of the cells from generation P3 to generation P10, may be any one of the cells from generation P4 to generation P10, and may be more specifically the cells from generation P3, generation P4, generation P5 or generation P6.

Any one of the above stem cells is a totipotent stem cell or a pluripotent stem cell.

Any one of the above stem cells is a human pluripotent stem cell or a human pluripotent stem cell.

The pluripotent stem cells are embryonic stem cells or induced pluripotent stem cells.

Any of the above stem cells is a human embryonic stem cell or an induced human pluripotent stem cell.

Illustratively, any of the stem cells described above is the H9 cell line.

Illustratively, any of the recombinant cells described above is a recombinant cell obtained by inserting nucleotide a after the 20 th nucleotide of the 4 th exon of SIRT7 gene in the genomic DNA of H9 cell line.

The gene editing is realized through a CRISPR/Cas9 system.

The target sequence of sgRNA of the CRISPR/Cas9 system is located at exon 4 of SIRT7 gene.

The target sequence of sgRNA of the CRISPR/Cas9 system is shown as sequence 1 in the sequence table.

The sgRNA of the CRISPR/Cas9 system is shown as a sequence 2 in a sequence table.

In any of the above methods, reducing the level of the SIRT7 protein in the stem cell and/or reducing the activity of the SIRT7 protein in the stem cell may be accomplished by any of the following means: inhibit the synthesis of the SIRT7 protein, inhibit the function of the SIRT7 protein and promote the degradation of the SIRT7 protein.

In any of the above methods, inhibiting the expression of a gene encoding SIRT7 protein in a stem cell can be achieved by any of the following means: RNA interference, gene silencing, gene knockout, or gene mutation.

In any of the above methods, reducing the level of a SIRT7 protein in a stem cell and/or reducing the activity of a SIRT7 protein in a stem cell may be accomplished by: adding a SIRT7 protein inhibitor to the stem cells. The SIRT7 protein inhibitor can be a protein, polypeptide or small molecule compound for inhibiting the synthesis of SIRT7 protein, or inhibiting the function of SIRT7 protein or promoting the degradation of SIRT7 protein.

In any of the above methods, inhibiting the expression of a gene encoding SIRT7 protein in a stem cell can be achieved by: adding RNA which can interfere the expression of the gene coding for the SIRT7 protein into the stem cell or adding a substance for knocking out or mutating the gene coding for the SIRT7 protein into the stem cell.

In any of the above methods, inhibiting the expression of a gene encoding SIRT7 protein in a stem cell can be achieved by: knocking out SIRT7 gene in stem cell.

The invention also provides a cell model prepared by any one of the methods.

The invention also protects any one of the following X1) -X4):

x1) a system for constructing the cell model consisting of a gRNA targeting the SIRT7 gene and a Cas9 nuclease;

x2) a system for constructing the cell model consisting of a vector expressing a gRNA targeting the SIRT7 gene and a vector expressing Cas9 nuclease;

x3) a system for constructing the cell model consisting of a gRNA targeting exon 4 of the SIRT7 gene and a Cas9 nuclease;

x4) for constructing the cell model, consisting of a vector expressing gRNA targeting exon 4 of SIRT7 gene and a vector expressing Cas9 nuclease.

In any of the above systems, the target sequence of the gRNA is shown as sequence 1 in the sequence listing.

In any of the above systems, the gRNA is represented by sequence 2 in the sequence table.

The carrier of gRNA for expressing the target SIRT7 gene is specifically shown as a sequence 3 in a sequence table.

The carrier for expressing the Cas9 nuclease is pCAG-1BPN L S-Cas9-1BPN L S-2 AGFP.

The system is specifically a product, and the product is specifically a kit.

The invention also protects the application of the cell model in any one of the following Y1) -Y7):

y1) as a model for cells with reduced proliferative capacity and/or in vivo retention;

y2) as a cell model for SIRT7 loss of function;

y3) as a cellular model for accelerated aging progression;

y4) for screening for substances which influence the expression of retrotransposons in cells and/or influence the proliferative capacity of cells and/or influence the persistence capacity in cells and/or influence the senescence process of cells;

y5) in the preparation of products; the product functions to screen for substances that affect the expression of cellular retrotransposons and/or that affect the proliferative capacity of cells and/or that affect the persistence capacity of cells in the body and/or that affect the senescence process of cells;

y6) for screening substances which inhibit the expression of retrotransposons and/or promote the proliferation of cells and/or promote the persistence of cells in vivo and/or delay the senescence of cells;

y7) in the preparation of products; the product has the function of screening substances for inhibiting the expression of retrotransposons and/or promoting the proliferation of cells and/or promoting the retention of cells in vivo and/or delaying the senescence of cells.

Y1) -Y7), any of the cells may be a mesenchymal stem cell, may be a human mesenchymal stem cell, may be any of the cell models (mesenchymal stem cell models) described above.

Y1) -Y7), the substance may be a drug and/or a natural organic substance and/or a small molecule compound and/or a toxic molecule.

The SIRT7 gene is a gene coding for SIRT7 protein.

Any one of the SIRT7 proteins is a human SIRT7 protein.

Illustratively, the human SIRT7 protein is (j1), (j2) or (j3) as follows:

(j1) protein shown in a sequence 4 in a sequence table;

(j2) the protein shown in the sequence 4 in the sequence table is subjected to substitution and/or deletion and/or addition of one or more amino acid residues, and has the same function as the protein and is derived from the protein;

(j3) a protein derived from a human and having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more identity to and the same function as the amino acid sequence defined in (j 1).

The encoding gene of the SIRT7 gene or the SIRT7 protein is specifically (k1) or (k2) or (k3) or (k 4):

(k1) the coding region is a DNA molecule shown as a sequence 6 in a sequence table;

(k2) DNA molecule shown in sequence 6 in the sequence table;

(k3) DNA molecule shown in sequence 5 in the sequence table;

(k4) a DNA molecule derived from a human and having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% or more identity to (k1) or (k2) or (k3) and encoding said protein.

Any of the retrotransposons described above may specifically be L INE 1.

The exon 4 of any one of the SIRT7 genes is shown as the 2473-2543 th nucleotide in the sequence 5 of the sequence table.

The research on the aging process of the SIRT 3634 by taking the mesenchymal stem cells as a model is helpful for people to further understand the aging process, the research on the scientific research on the function of SIRT7 for regulating the homeostasis of the mesenchymal stem cells is unknown, the population aging is a social problem which is increasingly severe in China and even all over, the research on the SIRT7 by taking the mesenchymal stem cells as a model is helpful for people to further understand the aging process, the research on the reverse transcription process of RNA (L INE1) can effectively inhibit the aging process caused by the accumulation of reverse transcription transposon (L INE1) in the stem cells, the research on the next clinical research on the human treatment of the aging related diseases can provide possibility for people to understand the physiological aging regulation mechanism of the human, and the research on the aging process of the SIRT7 obtained by editing the gene can provide the scientific application of the aging process, and the research on the aging process of the aging related diseases of the AIDS is not only for the research on the aging process of AIDS 593.

The mesenchymal stem cell model with SIRT7 function loss has the advantages of weakened cell proliferation capacity, accelerated degeneration speed, weakened in-vivo retention capacity and heterochromatin loss, and increased L INE1 transcription and expression, can be used for researching the relation between L INE1 transcription and expression and cell senescence, can be used for screening toxic molecules for delaying senescence of cells, and can be used for cell treatment as related materials with weakened proliferation and in-vivo retention capacity.

Drawings

FIG. 1 shows the results of example 1.

FIG. 2 shows the results of example 2.

FIG. 3 shows the results of example 3.

FIG. 4 shows the results of example 4.

FIG. 5 shows the results of example 5.

FIG. 6 shows the results of example 6.

Detailed Description

The following examples are provided to facilitate a better understanding of the present invention, but are not intended to limit the present invention, and the experimental methods in the following examples are all conventional methods, unless otherwise specified, and the test materials used in the following examples are all purchased from conventional biochemical reagent stores, and the quantitative tests in the following examples are all set up in triplicate, and the results are averaged, 3TC is collectively referred to as lamivudine (L amidovudine), Sigma-Aldrich, L1295-50 mg. matrigel, Invitrogen Inc., fluorescein FITC labeled anti-human cell surface recognition molecule CD90 antibody, BD Biosciences, Cat 555595, fluorescein PE labeled anti-human cell surface recognition molecule CD73 antibody, BD Biosciences, Cat 550257, fluorescein APC labeled anti-human cell surface recognition molecule CD105 antibody, eBiosciences, Cat 17-1057-42.

Cas9 expression vector pCAG-1BPN L S-Cas9-1BPN L S-2AGFP, product of Addgene, cat #87109, H9 cell line (human embryonic stem cell line) product of WiCell, cat # WA09(H9) -D L-7.

Unless otherwise specified, the mesenchymal stem cell passaging methods (each passage) in the examples were: cells were harvested, trypsinized, and cultured for 4 days with MSC medium (with new MSC medium every 2 days).

Unless otherwise specified, the cell culture conditions in the examples were all 37 ℃ and 5% CO₂。

The pluripotent stem cell medium (CDF12 medium) contained 0.1mM nonessential amino acids (Invitrogen, Cat. No. 11140-^TMDipeptide (Invitrogen, Cat. No. 35050-nVitrogen, cat # N10828-028), 1g/100ml penicillin/streptomycin (Invitrogen, cat # 15070-063), 55. mu.M β -mercaptoethanol (Invitrogen, cat # 21985-023) and 10ng/ml DMEM/F12 medium of human FGF2(Joint Protein Central), DMEM/F12 medium: Invitrogen, cat # 11320-033.

Mesenchymal stem cell medium (MSC medium) is an MEM medium containing 10% (by volume) fetal bovine serum (Gemcell, Inc., cat # 100- "500), 0.1mM non-essential amino acids (Invitrogen, cat # 11140-" 050), 1mM GlutamAXTM dipeptide (Invitrogen, cat # 35050- "061), 1g/100ml penicillin/streptomycin (Invitrogen, cat # 15070-" 063), and 1ng/ml human FGF2(Joint Protein Central Co.). MEM medium (product of Invitrogen, cat # 12571071).

The mesenchymal stem cell-inducing differentiation medium (MSC-inducing differentiation medium) is a MEM medium (product of Invitrogen corporation, Cat.: 12571071) containing 10% by volume of fetal bovine serum (Gemcell, Cat.: 100-500), 0.1mM of nonessential amino acids (Invitrogen, Cat.: 11140-050), 1mM of GlutaMAXTM dipeptide (Invitrogen, Cat.: 35050-061), 1g/100ml of penicillin/streptomycin (Invitrogen, Cat.: 15070-063), 10ng/ml of human FGF2(Joint Protein Central Co.), 5ng/m L TGF- β (Humanzyme).

Example 1 preparation of SIRT7 loss-of-function human embryonic Stem cells

First, construction of SIRT7-gRNA-mCherry

gRNA sequences were designed that target exon 4 of the human SIRT7 gene.

Target sequence of gRNA sequence (sequence 1 of sequence listing): 5'-CAGCGTCTATCCCAGACTAC-3' are provided.

Preparing SIRT7-gRNA-mCherry plasmid. The complete sequence of the SIRT7-gRNA-mCherry plasmid is shown as a sequence 3 in a sequence table. The gRNA shown in the sequence 2 of the sequence table is expressed by the SIRT7-gRNA-mCherry plasmid.

II, SIRT7^-/-Acquisition of hESC cell lines

1. The H9 cell line proliferating in log phase was taken, washed with PBS buffer, then digested with Tryp L E Express (Invitrogen corporation, cat. No. 12604021) for 5-10min, and then gently blown into single cells.

2.5 × 10⁶Each H9 single cell, 14. mu.g Cas9 expression vector and 7. mu.g SIRT7-gRNA-mCherry plasmid were mixed, and then brought to 100. mu. L using opti-MEM (life technology), and transferred to a 4D electric transfer apparatus (L naza, cat # 4D-Nucleofector)^TMSystem) to perform electrical transfer.

3. The cells after electroporation were added to a Matrigel-coated plate and cultured with CDF12 medium for 24 hours, and then observed for green fluorescence and red fluorescence with a fluorescence microscope. If the proportion of double positive cells is more than 1% (i.e., the proportion of cells which emit green fluorescence and red fluorescence simultaneously is more than 1%), the next step can be performed.

4. After step 3 was completed, the cells were collected by Tryp L E digestion, and then GFP and mCherry double positive cells were sorted by flow cytometry.

5. And (4) placing the double-positive cells obtained by sorting in the step (4) into the holes of a six-hole plate which is paved with mouse embryo fibroblasts subjected to mitomycin inactivation treatment in advance, and culturing for about 14 days by using a CDF12 culture medium, so that small clones formed by single cells can be observed.

6. Each single cell clone was subcultured (using CDF12 medium for 7 days), and the subcultured cells were identified by PCR. The PCR identification method comprises the following steps: extracting genome DNA, carrying out PCR amplification by using a primer pair consisting of SIRT7-F and SIRT7-R, and then recovering and sequencing PCR amplification products.

SIRT7-F：5’-GACAGTCGCCTGGACCCACCT-3’；

SIRT7-R：5’-ACCCAGGGCTCTTACCAGCTT-3’。

Screening to obtain a single cell clone, wherein the sequencing region has homozygous mutation in the form of nucleotide A inserted after the 20 th nucleotide of exon 4 of SIRT7 gene (the nucleotide and the first two nucleotides together form a stop codon to terminate in advance), and the single cell clone is named SIRT7^-/-hESC cells.

SIRT7^-/-hESC cells are human embryonic stem cells with loss of function of SIRT 7.

Mixing SIRT7^-/-The cell obtained by subculturing hESC cell is named SIRT7^-/-hESC cell line.

H9 cell line (with SIRT 7)^+/+Representation) and SIRT7^-/-hESC cell line (with SIRT 7)^-/-Representation) are shown in FIG. 1A.

Third, Western blotting identification of SIRT7 protein

Test cells: h9 cell line (with SIRT 7)^+/+Representation) or SIRT7^-/-hESC cell line (with SIRT 7)^-/-Representation).

Total protein of test cells was extracted and subjected to Western blotting to detect SIRT7 protein as a target, SIRT7 antibody as a primary antibody (anti-SIRT7, rabbit-derived, product of Cell Signaling Technology, cat #5360S), HRP-labeled goat anti-rabbit antibody as a secondary antibody (product of Medtronic bridge, cat # ZB-2301), β -Tubulin as an internal reference, mouse anti- β -Tubulin antibody as a primary antibody (product of Santa cruz, cat # sc-5274), and HRP-labeled goat anti-mouse antibody as a secondary antibody (product of Medtronic bridge, cat # ZB-2305), the results of which are shown in FIG. 1 B.SIRT7^-/-The hESC cell line did not detect expression of SIRT7 protein; expression of the SIRT7 protein (molecular weight approximately 44kDa) was detected by the H9 cell line.

Fourth, immunofluorescence assay

Immunofluorescence was performed to detect the levels of proteins expressed by pluripotency genes (OCT4, SOX2, NANOG) in test cells. The antibodies used for immunofluorescence identification were as follows: anti-NANOG (Abcam, ab21624), anti-OCT4(Santa Cruz, sc5279), anti-SOX2(Santa Cruz, sc 17320). The results are shown in C of FIG. 1. Both cells tested normally expressed the pluripotency sternness gene (OCT4, SOX2, NANOG).

Example 2 preparation and identification of SIRT7 function-lost human mesenchymal Stem cells

Preparation of human mesenchymal stem cells with SIRT7 loss function

1. Mixing SIRT7^-/-hESC cell lineAnd performing embryoid body differentiation to obtain Embryoid Body (EB).

2. The embryoid bodies obtained in step 1 were inoculated into a Matrigel-coated 6-well plate and cultured for 10 days with an MSC-induced differentiation medium (replacement of the MSC-induced differentiation medium with a new one every 2 days), and the appearance of fibroblasts was observed.

3. After completion of step 2, the cells were collected, trypsinized, and then inoculated into MSC medium and cultured for 10 days (replacement of new MSC medium every 2 days).

4. After step 3 is completed, sorting the cells positive for CD73, CD90 and CD105 (see A in figure 2) by flow cytometry, namely the human mesenchymal stem cells with SIRT7 loss of function, which are also called SIRT7^-/-hMSC cells (passage P1).

5. The P1 generation SIRT7^-/-The hMSC cells are frozen and preserved after being passaged once and are recovered when in use, namely the P2 generation SIRT7^-/-hMSC cells.

II, preparing SIRT7 by in vitro directional differentiation of H9 cell line^+/+hMSC

1. H9 cell line was subjected to embryoid body differentiation to obtain Embryoid Body (EB).

2. The same as step one, 2.

3. The same as step one, 3.

4. After step 3 is completed, sorting cells positive for CD73, CD90 and CD105 (see A in FIG. 2) by flow cytometry to obtain control human mesenchymal stem cells, also called SIRT7^+/+hMSC cells (passage P1).

5. The P1 generation SIRT7^+/+The hMSC cells are frozen and preserved after being passaged once and are recovered when in use, namely the P2 generation SIRT7^+/+hMSC cells.

Third, phenotypic identification

1. Western blotting detection of SIRT7 protein abundance

Test cells: SIRT7^+/+hMSC cells (P2 generation) or SIRT7^-/-hMSC cells (passage P2).

The procedure is as in step three of example 1.

The results are shown in B of FIG. 2. SIRT7^-/-The hMSC cells can not detect the expression of SIRT7 protein; SIRT7^+/+The SIRT7 protein is detected by the hMSC(molecular weight is about 44 kDa).

2. Determination of proliferation potency

The P2 test cells were serially passaged 11 times. The cells obtained from passage 1 are P3 generation cells, the cells obtained from passage 2 are P4 generation cells, and so on, and the cells obtained from passage 11 are P13 generation cells.

The cell growth factor per generation was calculated as the number of cells at the end of each generation/the number of cells at the start of each generation.

Log (cumulative proliferation multiple) of P3 generation cells₂(P3 fold proliferation of cells).

Log (cumulative proliferation multiple) of P4 generation cells₂(P3 fold cell proliferation) + log₂(P4 fold proliferation of cells).

Log (cumulative proliferation multiple) of P5 generation cells₂(P3 fold cell proliferation) + log₂(P4 fold cell proliferation) + log₂(P5 fold proliferation of cells).

And so on.

The results are shown in C of FIG. 2. When SIRT7^-/-SIRT7 when the hMSC proliferation rate is slowed down^+/+The hMSC can still maintain a faster proliferation rate. SIRT7^-/-The generation of hMSC reaching growth retardation is P10 generation, SIRT7 generation^+/+hmscs did not reach growth arrest until P13 generation. The results show that: and SIRT7^+/+SIRT7 in comparison with hMSC cells^-/-hMSC proliferative capacity is diminished.

3. SA- β -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 (senesce-associated beta-galactosidase) activity during senescence when X-Gal is used as a substrate, the cells produce a dark blue product under the catalysis of the senescence-specific β -galactosidase.

Respectively using SIRT7^+/+Passage cell of hMSC cell and SIRT7^-/-The passage cells of the hMSC cells are the test cells. The early generation cells are P3 generation cells(in EP.) the late cell generation was P8 cell generation (in L P).

The test cells were stained with SA- β -Gal, photographed and counted for the proportion of SA- β -Gal positive cells.

The results are shown in D of FIG. 2. Early generation cells, SIRT7^+/+hMSC and SIRT7^-/-The proportion of SA- β -Gal staining positive cells in hMSC is not obviously different, and the late generation cells are different from SIRT7^+/+SIRT7 compared to hMSC^-/-The proportion of SA- β -Gal staining positive cells of hMSC was significantly higher (P)<0.01). The results show that SIRT7 appears with cell passage^-/-Senescence progression of hMSCs compared to SIRT7^+/+hmscs accelerated significantly.

4. Determination of Retention Capacity in mice

A lentiviral vector over expressing luciferase, which is called luc lentiviral vector for short. Reference documents: SIRT6 safety and design human sensory cells from oxidative stress by immunological activity NRF2.Pan et al,. Cell research (2016)26: 190-; namely "luciferase (control) -expressed vector" in the literature.

Lentiviral packaging plasmid psPAX, abbreviated plasmid psPAX, Adgene, cat # 12260 lentiviral packaging plasmid pMD2.G, abbreviated plasmid pMD2.G, Adgene, cat # 12259.293T cells (human embryonic kidney cells): ATCC, CR L-3216.

Test cells: SIRT7^+/+hMSC cells (P6 generation) or SIRT7^-/-hMSC cells (passage P6).

The experimental animal is BA L B/c male nude mice, Beijing Wintonli Hua experimental animal technology Limited.

(1) Luciferase-labeled test cells were prepared.

① mu.g of luc lentiviral vector, 10. mu.g of plasmid psPAX and 5. mu.g of plasmid pMD.2G were co-transfected into 293T cells using L ipofectamine 3000 kit (Invitrogen), cultured for 48 hours, and then the supernatant was collected.

② centrifuging the supernatant obtained in step ① at 19200rpm at 4 deg.C for 2.5 hr, discarding the supernatant, and suspending and precipitating with MSC culture medium to obtain virus solution.

③ test cells (MOI 3btu/cell) were infected with the virus solution obtained in step ②, cultured for 3 days using MSC medium, the cells were collected, trypsinized, inoculated into new MSC medium and cultured for 3 days to obtain luciferase-labeled test cells.

(2) Transplanting cells into tibialis anterior of mice

Taking SIRT7 labeled by luciferase^+/+And (4) resuspending the hMSC by using PBS buffer solution to obtain cell fluid A. Luciferase-tagged SIRT7 in 100. mu.l of cytosolic A^+/+The content of hMSC is 1 × 10⁶And (4) respectively. Taking SIRT7 labeled by luciferase^-/-And (4) resuspending the hMSC by using PBS buffer solution to obtain cell fluid B. Luciferase-tagged SIRT7 in 100. mu.l of cytosol B^-/-The content of hMSC is 1 × 10⁶And (4) respectively.

Test animals were taken, 100. mu.l of a cell sap A was injected into the tibialis anterior muscle of each left leg, 100. mu.l of a cell sap B was injected into the tibialis anterior muscle of the right leg, and after 0, 3 and 5 days, mice were taken, 100. mu. l D-L uciferin (15mg/m L) (D-L uciferinfirely, podassium salt, GO L DBIO Co.) were intraperitoneally injected, and after gas anesthesia, analysis was performed using a small animal living body imaging system.

The photograph after 5 days is shown in the left panel of E of FIG. 2.

0. The relative fluorescence intensity after 3 and 5 days is shown in the right graph of FIG. 2E (average of five mice).

SIRT7^-/-The luciferase activity of hMSC (right leg) in tibialis anterior muscle is higher than SIRT7^+/+hMSC (left leg) is significantly attenuated in the tibialis anterior. The results show that the protein has the same effect as SIRT7^+/+SIRT7 compared to hMSC^-/-hMSC has increased in vivo degenerative rate and decreased retention ability.

Example 3 SIRT7^-/-Electron microscope detection of hMSC (Observation of heterochromatin distribution)

1. Test cells were collected, digested, and then washed with PBS buffer.

2. The cells were harvested, resuspended in 4% PFA in PBS buffer, and then left overnight at 4 ℃.

3. Cells were dehydrated with fractionated ethanol and infiltrated with low concentration resin HM 20.

4. Image acquisition was performed using an alcohol transmission electron microscope (FEI corporation) operating at 100 kv.

The photograph under the electron microscope is shown in FIG. 3. The results show that SIRT7^-/-heterochromatin loss from hmscs occurred.

Example 4 SIRT7^-/-L INE1 expression assay for hMSC

First, RT-qPCR detection

Respectively using SIRT7^+/+Passage cell of hMSC cell and SIRT7^-/-The early generation cells were P3 generation cells (indicated by EP), the late generation cells were P8 generation cells (indicated by L P).

1. Taking a test cell, extracting total RNA, and carrying out reverse transcription to obtain cDNA.

2. The cDNA obtained in step 1 was used as a template, GAPDH was used as an internal reference gene, and the transcription level of L INE1 was detected by RT-qPCR.

Primer pairs for detecting L INE1 were as follows:

LINE1-F：5'-CAGAGACACACATAGGCTCAAA-3'；

LINE1-R：5'-AATCTGGGTGCTCCTGTATTG-3'。

primer pairs for detecting GAPDH are as follows:

GAPDH-F：5'-TCGGAGTCAACGGATTTGGT-3'；

GAPDH-R：5'-TTGCCATGGGTGGAATCATA-3'。

the results are shown in A of FIG. 4. Compared with SIRT7^+/+hMSC，SIRT7^-/-L INE1 transcript levels were increased in hMSCs.

Second, Western blotting detection

Total protein of test cells was extracted, and Western blotting was performed to detect that the target protein was L INE1 ORF1p, primary antibody was L INE1 ORF1p antibody (anti-ORF1p, murine, Millipore Corp., cat # MABC1152), secondary antibody was HRP-labeled goat anti-mouse antibody (China fir bridge Corp., cat # ZB-2305), target protein was L INE1 ORF2p, primary antibody was L INE1 ORF2p antibody (anti-ORF2p, chicken-derived, Abcam Corp., cat # ab106004), secondary antibody was HRP-labeled goat anti-chicken antibody (Abcam Corp., cat # ab6877), secondary antibody was H3 was endogenous reference, primary antibody was H3 antibody (Abcam Corp., rabbit-derived, cat # ab1791), and rabbit anti-rabbit antibody (HRP-labeled goat anti-rabbit antibody; ZB-2301).

The results are shown in B of FIG. 4. Compared with SIRT7^+/+hMSC，SIRT7^-/-ORF1p and ORF2p are highly expressed in hMSC (ORF1 has a molecular weight of about 40kDa, ORF2p has a molecular weight of about 149 kDa).

The results of step one and step two show that SIRT7 is an inhibitor of L INE1 in hMSCs, compared to SIRT7^+/+hMSC，SIRT7^-/-L INE1 transcription and translation levels of hMSC are increased, and L INE1 transcription activity after SIRT7 function loss is probably an important reason for slow cell proliferation and accelerated aging process.

Example 5 SIRT7 after 3TC treatment^-/-L INE1 expression assay for hMSC cells

One, packet processing

First group (3TC group): SIRT7^-/-hMSC cells (passage P3) were inoculated into MSC medium containing 10 μ M3 TC and cultured for 4 days (the same medium was changed every 2 days); then collecting cells for trypsinization, inoculating to a new MSC medium containing 10. mu.M 3TC, and culturing for 4 days (changing the same medium every 2 days); the cells were then harvested for trypsinization, inoculated into fresh MSC medium containing 10. mu.M 3TC, and cultured for 4 days (replacement of fresh same medium every 2 days).

Second group (vehicle group): the MSC medium containing 10. mu.M 3TC was replaced with the MSC medium, and the same was used for the first group.

Second, RT-qPCR detection

1. After grouping treatment, cells are respectively collected, total RNA is extracted, and cDNA is obtained through reverse transcription.

2. And (3) detecting the transcription level of L INE1 by RT-qPCR by using the cDNA obtained in the step 1 as a template.

The procedure was the same as in 2 of step one of example 4.

The results are shown in A of FIG. 5. SIR compared to vehicle processingT7^-/-hMSC, 3TC treated SIRT7^-/-The transcriptional level of L INE1 was reduced for hMSCs.

Third, Western blotting detection

After the grouping treatment, the cells were collected separately as test cells.

The procedure is as in step two of example 4.

The results are shown in B of FIG. 5. Compared with the SIRT7 treated by vehicle^-/-hMSC, 3TC treated SIRT7^-/-The level of translation of L INE1 by hMSC was reduced.

Example 6 SIRT7 after 3TC treatment^-/-Senescence phenotype assay for hMSC cells

One, packet processing

Same as step one of example 5.

Second, monoclonal formation experiment for detecting cell proliferation ability

After the grouping treatment, the cells were collected separately and subjected to a single colony formation experiment.

The method of the monoclonal formation experiment was as follows:

1. 2000 cells were plated in six-well plates and cultured with MSC medium for 10 days (with new MSC medium every 2 days).

2. After completion of step 1, fixation with 4% PFA was performed for 30min, followed by washing with PBS buffer.

3. Dyeing for 30min by using a crystal violet dye solution diluted by 10 times, and then washing and airing by using clear water.

4. The scanner scans and the software image J calculates the gray value.

The results are shown in A of FIG. 6. 3TC treated SIRT7^-/-The hMSC had a greater crystal violet staining depth than the vehicle treated SIRT7^-/-hMSC. The results show that: 3TC treated SIRT7^-/-hMSC compared to vehicle-treated SIRT7^-/-The proliferation capacity of hMSC is accelerated.

Third, SA- β -Gal staining

After the grouping treatment, the cells were collected, stained with SA- β -Gal, photographed, and counted for the SA- β -Gal positive cell ratio.

The results are shown in B of FIG. 6. Compared with vehicle processingSIRT7^-/-hMSC, 3TC treated SIRT7^-/-The proportion of SA- β -Gal staining positive cells of hMSC is obviously reduced (P)<0.001). The result shows that the 3TC can effectively delay SIRT7^-/-Senescence progression of hmscs.

SEQUENCE LISTING

<110> institute of animal research of Chinese academy of sciences

Application of <120> lamivudine in delaying senescence and preparation of cell model for screening anti-senescence drugs

<130>GNCYX201039

<160>6

<170>PatentIn version 3.5

<210>1

<211>20

<212>DNA

<213>Homo sapiens

<400>1

cagcgtctat cccagactac 20

<210>2

<211>100

<212>RNA

<213>Artificial sequence

<400>2

cagcgucuau cccagacuac guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60

cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100

<210>3

<211>5695

<212>DNA

<213>Artificial sequence

<400>3

caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc 60

cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta 120

taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg 180

ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc 240

tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac 300

gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac 360

ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg 420

aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga 480

agacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta 540

gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc 600

agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg 660

acgctcagtg gaacgaaaac tcacgttaag ggattattgg tcatgagatt atcaaaaagg 720

atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat 780

gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc 840

tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg 900

gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct 960

ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca 1020

actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg 1080

ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg 1140

tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc 1200

cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag 1260

ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg 1320

ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag 1380

tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat 1440

agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg 1500

atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca 1560

gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca 1620

aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat 1680

tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag 1740

aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgggtcgaca 1800

ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc atagcccata 1860

tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga 1920

cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt 1980

ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt 2040

gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca 2100

ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt 2160

catcgctatt accatggtcg aggtgagccc cacgttctgc ttcactctcc ccatctcccc 2220

cccctcccca cccccaattt tgtatttatt tattttttaa ttattttgtg cagcgatggg 2280

ggcggggggg gggggggggc gcgcgccagg cggggcgggg cggggcgagg ggcggggcgg 2340

ggcgaggcgg agaggtgcgg cggcagccaa tcagagcggc gcgctccgaa agtttccttt 2400

tatggcgagg cggcggcggc ggcggcccta taaaaagcga agcgcgcggc gggcgggagt 2460

cgctgcgttg ccttcgcccc gtgccccgct ccgcgccgcc tcgcgccgcc cgccccggct 2520

ctgactgacc gcgttactcc cacaggtgag cgggcgggac ggcccttctc ctccgggctg 2580

taattagcgc ttggtttaat gacggctcgt ttcttttctg tggctgcgtg aaagccttaa 2640

agggctccgg gagggccctt tgtgcggggg ggagcggctc ggggggtgcg tgcgtgtgtg 2700

tgtgcgtggg gagcgccgcg tgcggcccgc gctgcccggc ggctgtgagc gctgcgggcg 2760

cggcgcgggg ctttgtgcgc tccgcgtgtg cgcgagggga gcgcggccgg gggcggtgcc 2820

ccgcggtgcg ggggggctgc gaggggaaca aaggctgcgt gcggggtgtg tgcgtggggg 2880

ggtgagcagg gggtgtgggc gcggcggtcg ggctgtaacc cccccctgca cccccctccc 2940

cgagttgctg agcacggccc ggcttcgggt gcggggctcc gtacggggcg tggcgcgggg 3000

ctcgccgtgc cgggcggggg gtggcggcag gtgggggtgc cgggcggggc ggggccgcct 3060

cgggccgggg agggctcggg ggaggggcgc ggcggccccc ggagcgccgg cggctgtcga 3120

ggcgcggcga gccgcagcca ttgcctttta tggtaatcgt gcgagagggc gcagggactt 3180

cctttgtccc aaatctgtgc ggagccgaaa tctgggaggc gccgccgcac cccctctagc 3240

gggcgcgggg cgaagcggtg cggcgccggc aggaaggaaa tgggcgggga gggccttcgt 3300

gcgtcgccgc gccgccgtcc ccttctccct ctccagcctc ggggctgtcc gcggggggac 3360

ggctgccttc gggggggacg gggcagggcg gggttcggct tctggcgtgt gaccggcggc 3420

tctagagcct ctgctaacca tgttcatgcc ttcttctttt tcctacagct cctgggcaac 3480

gtgctggtta ttgtgctgtc tcatcatttt ggcaaagaat tgctcgagat gtatccgtat 3540

gatgtgccgg attatgcggt gagcaagggc gaggaggata acatggccat catcaaggag 3600

ttcatgcgct tcaaggtgca catggagggc tccgtgaacg gccacgagtt cgagatcgag 3660

ggcgagggcg agggccgccc ctacgagggc acccagaccg ccaagctgaa ggtgaccaag 3720

ggtggccccc tgcccttcgc ctgggacatc ctgtcccctc agttcatgta cggctccaag 3780

gcctacgtga agcaccccgc cgacatcccc gactacttga agctgtcctt ccccgagggc 3840

ttcaagtggg agcgcgtgat gaacttcgag gacggcggcg tggtgaccgt gacccaggac 3900

tcctccctgc aggacggcga gttcatctac aaggtgaagc tgcgcggcac caacttcccc 3960

tccgacggcc ccgtaatgca gaagaagacc atgggctggg aggcctcctc cgagcggatg 4020

taccccgagg acggcgccct gaagggcgag atcaagcaga ggctgaagct gaaggacggc 4080

ggccactacg acgctgaggt caagaccacc tacaaggcca agaagcccgt gcagctgccc 4140

ggcgcctaca acgtcaacat caagttggac atcacctccc acaacgagga ctacaccatc 4200

gtggaacagt acgaacgcgc cgagggccgc cactccaccg gcggcatgga cgagctgtac 4260

aagtaagcgg ccgcaattca ctcctcaggt gcaggctgcc tatcagaagg tggtggctgg 4320

tgtggccaat gccctggctc acaaatacca ctgagatctt tttccctctg ccaaaaatta 4380

tggggacatc atgaagcccc ttgagcatct gacttctggc taataaagga aatttatttt 4440

cattgcaata gtgtgttgga attttttgtg tctatcactc ggaaggacat atgggagggc 4500

aaatcattta aaacatcaga atgagtattt ggtttagagt ttggcaacat atgcccatat 4560

gctggctgcc atgaacaaag gttggctata aagaggtcat cagtatatga aacagccccc 4620

tgctgtccat tccttattcc atagaaaagc cttgacttga ggttagattt tttttatatt 4680

ttgttttgtg ttattttttt ctttaacatc cctaaaattt tccttacatg ttttactagc 4740

cagatttttc ctcctctcct gactactccc agtcatagct gtccctcttc tcttatggag 4800

atccctcgac ctgcaccgtc gaccagctgg tcgacggtgc accgtcgacc agcttggcgt 4860

aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca 4920

tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat 4980

taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagcggatct 5040

gtacaaaaaa gcaggcttta aaggaaccaa ttcagtcgac tggatccggt accaaggtcg 5100

ggcaggaaga gggcctattt cccatgattc cttcatattt gcatatacga tacaaggctg 5160

ttagagagat aattagaatt aatttgactg taaacacaaa gatattagta caaaatacgt 5220

gacgtagaaa gtaataattt cttgggtagt ttgcagtttt aaaattatgt tttaaaatgg 5280

actatcatat gcttaccgta acttgaaagt atttcgattt cttggcttta tatatcttgt 5340

ggaaaggacg aaacacccag cgtctatccc agactacgtt ttagagctag aaatagcaag 5400

ttaaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttt 5460

ctagacccag ctttcttgta caaagttggc attagatccg ctgcattaat gaatcggcca 5520

acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc 5580

gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg 5640

gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccag 5695

<210>4

<211>400

<212>PRT

<213>Homo sapiens

<400>4

Met Ala Ala Gly Gly Leu Ser Arg Ser Glu Arg Lys Ala Ala Glu Arg

1 5 10 15

Val Arg Arg Leu Arg Glu Glu Gln Gln Arg Glu Arg Leu Arg Gln Val

20 25 30

Ser Arg Ile Leu Arg Lys Ala Ala Ala Glu Arg Ser Ala Glu Glu Gly

35 40 45

Arg Leu Leu Ala Glu Ser Ala Asp Leu Val Thr Glu Leu Gln Gly Arg

50 55 60

Ser Arg Arg Arg Glu Gly Leu Lys Arg Arg Gln Glu Glu Val Cys Asp

65 70 75 80

Asp Pro Glu Glu Leu Arg Gly Lys Val Arg Glu Leu Ala Ser Ala Val

85 90 95

Arg Asn Ala Lys Tyr Leu Val Val Tyr Thr Gly Ala Gly Ile Ser Thr

100 105 110

Ala Ala Ser Ile Pro Asp Tyr Arg Gly Pro Asn Gly Val Trp Thr Leu

115 120 125

Leu Gln Lys Gly Arg Ser Val Ser Ala Ala Asp Leu Ser Glu Ala Glu

130 135 140

Pro Thr Leu Thr His Met Ser Ile Thr Arg Leu His Glu Gln Lys Leu

145 150 155 160

Val Gln His Val Val Ser Gln Asn Cys Asp Gly Leu His Leu Arg Ser

165 170 175

Gly Leu Pro Arg Thr Ala Ile Ser Glu Leu His Gly Asn Met Tyr Ile

180 185 190

Glu Val CysThr Ser Cys Val Pro Asn Arg Glu Tyr Val Arg Val Phe

195 200 205

Asp Val Thr Glu Arg Thr Ala Leu His Arg His Gln Thr Gly Arg Thr

210 215 220

Cys His Lys Cys Gly Thr Gln Leu Arg Asp Thr Ile Val His Phe Gly

225 230 235 240

Glu Arg Gly Thr Leu Gly Gln Pro Leu Asn Trp Glu Ala Ala Thr Glu

245 250 255

Ala Ala Ser Arg Ala Asp Thr Ile Leu Cys Leu Gly Ser Ser Leu Lys

260 265 270

Val Leu Lys Lys Tyr Pro Arg Leu Trp Cys Met Thr Lys Pro Pro Ser

275 280 285

Arg Arg Pro Lys Leu Tyr Ile Val Asn Leu Gln Trp Thr Pro Lys Asp

290 295 300

Asp Trp Ala Ala Leu Lys Leu His Gly Lys Cys Asp Asp Val Met Arg

305 310 315 320

Leu Leu Met Ala Glu Leu Gly Leu Glu Ile Pro Ala Tyr Ser Arg Trp

325 330 335

Gln Asp Pro Ile Phe Ser Leu Ala Thr Pro Leu Arg Ala Gly Glu Glu

340 345 350

Gly Ser His Ser ArgLys Ser Leu Cys Arg Ser Arg Glu Glu Ala Pro

355 360 365

Pro Gly Asp Arg Gly Ala Pro Leu Ser Ser Ala Pro Ile Leu Gly Gly

370 375 380

Trp Phe Gly Arg Gly Cys Thr Lys Arg Thr Lys Arg Lys Lys Val Thr

385 390 395 400

<210>5

<211>6215

<212>DNA

<213>Homo sapiens

<400>5

agagcaggtc tccaggggag cgatggcagc cgggggtctg agccgctccg agcgcaaagc 60

ggcggagcgg gtccggaggt tgcgggagga gcagcagagg gagcgcctcc gccaggtacg 120

ccgccgccgc tccccggccc ggccatgccc ggcccgcgcc gccgctcacc gtccgcctgc 180

ccgcaggtgt cgcgcatcct gaggaaggcg gcggcggagc gcagcgccga ggagggccgg 240

ctgctggccg agagcgcgga cctggtaacg gagctgcagg gccggagccg gcggcgcgag 300

ggcctgaagc ggcggcagga ggaggcgagt tccgcgtgcg gcgcgcgggc gcccccggtt 360

tcgggagcag ctggggcgac gggcggtccc gggtggggcg gcccggggcg gtgaccaccc 420

tggcgtcttg gcaggtgtgc gacgacccgg aggagctgcg ggggaaggtc cgggagctgg 480

ccagcgccgt ccggaacgcc aaatacttgg tcgtctacac aggcgcggga atcagcacgg 540

tagggaggga ggcggaggcg taccccagga cggagtatga gctccagtaa tcgcgaaaaa 600

ctcgccttta aagcagctct aaggtttttt ctcttaaaga aacgaaatga ccaaaactta 660

cctaaggtaa acgcttttta aacgcttggc ctctgtgtta cagccagtta aaaaaaacaa 720

ggagtagaga tacgaatggg gtgtagtagc cgactgctcg caggcacccc caggttatgt 780

ggacagagct aagcccaaag ttgtgatttt ccactctgtt ctgtccatgt cgagggaaga 840

taagtagaaa gtgacacagt aagagccaga atacaccagg tgaaggagag aattgcattg 900

tgttttgaga agtttcactg acaagttatc ctgggctgtg ggacatcact agctttgaaa 960

gtgtagctgg cacctcgtcc atctaatttg atgggtgtgt gtggggtgtt gggcacgcgt 1020

cggcctagca gatctgaacc caggtgattt ctgttctcag gaagctttta ggtgacaagg 1080

atcaggcatg tgaacaaata accatactgt aaagctggct gtgctgggtc gctagagcaa 1140

actcagacac acactctgcg ctcttggagt tgggaaaccc acctgcgttg gctttttgtg 1200

ggaggtggcc ttgattgggc cttgaaggat gggtgagatt tacagaaggt tggaatgagg 1260

cactccaagc aaagaacagc agaggctcag cagcaagaat gcaaaaaggg agttcactac 1320

tgactcaaat acccggagcc ctggggttta gtctcctccc tccacaagtc acatgtaaaa 1380

gtccagtcat gctgggcgcg gtggctcatg cctataatcc tagcactttg ggaggccgag 1440

gtgggcggat catgaggtca gtagttcaag aacaacctgg ccaacacggt gaaaccctgt 1500

ctctactaaa aatacaaaaa ttagtcgggc atggtggcgg gcacctgcaa tcccagctac 1560

tcgagaggct gaggcaggag aatcacttga aactggaagg cggaggttgc agtgagccga 1620

gatcatgcca ctgcattcca gcctgggcga aagagctaaa ctccgtctct caaaaaaaaa 1680

aaaaaaaaaa aaagtccagt catgtaatta tgtaacagtc acgtgacctg ttatggaact1740

tccaatggca actaaaagca catgcagcta gtggatttca tcggagtgtt tgaggttccc 1800

gtcttgaatg tgactgtcgg aactactgtc cgggggggtg gtgcattttt ctgagtttaa 1860

gcaggagtcg ggagtcccca aaagggaaca caagacacct tgatcctggc atatcttgtg 1920

tgccctctgt gggcctcagt ttgtttcaca ggattgaaaa cctgggaagt tagatgctca 1980

tctcatctga agaagttgtt ctgcctttgt taaggtggag cgggaatagt cagcactggg 2040

acatgagaat ggacagtcgc ctggacccac ctagggattc accatttgct aaatgtgtga 2100

gctgtgggct cggccctggg ggcactttag gaacatgact agtcttcccc tgcagtgtgg 2160

aggacacatg tgccacagag cccagctttg tgctcggtgc cagagaggct tccggaggca 2220

ggcagggctg cgtgcagcct ggaaggatga gccaggccag gcgggaaacg gaagtccagg 2280

tagaagggag gagccgaatt ggggtacact ccatatgggc tcaggcaggt cagcctgtgg 2340

aatgaataga ggccaacatg caggccagcc cggaatgcgg caggagtgac agtggctttc 2400

cgtttctggg aattctgcca gtacctacag tggtgccttt tgacttggct tacctttttt 2460

ctcgacatgc aggcagcgtc tatcccagac taccggggcc ctaatggagt gtggacactg 2520

cttcagaaag ggagaagcgt taggtaagcg ggccaggcat ggcctcccac ataggctggg 2580

cagcggcagc acgggcctga gctccagctc tcctcacctt gccttccttt ctgcctggca 2640

gtgctgccga cctgagcgag gccgagccaa ccctcaccca catgagcatc acccgtctgc 2700

atgagcagaa gctggtaaga gccctgggtg gctggtacac ttgccaggga ccaggcagag 2760

caccttggtg cccagtgggc aactaactgc acccgccctc tgtctgccag ttgactccca 2820

tgatgagcac ccaccaagcg ggttaggccg cgggtttgat cctcctgtgc ttgactctcc 2880

aggacagaag gggagctccc cctctgaacc atcccatccg cagccagcct cagcctcaga 2940

gctgctggtg gcctttcccc tttgtgattc ccttgcattt ttctgtggac ctcagaagcc 3000

atcctagtca caggggaggc ttataggaca tctctgggga ccttctgctg aacaccctca 3060

accaatgggg tgtagtggtt gatctgccta ggtccccagg gacctgaaat gtcatgaccc 3120

aagagagcat ggatctgggg cagagtggcc cttgccaaac cccgagccac ttcccaacct 3180

tgccgggacg gtgcaacctc ttgccctccc agccactcca ggtgcatcag ggctggagga 3240

aggacagccc ctccccacca caggccctct tgactcctgg tggttggacc tgttgtgtgt 3300

tttactttct aaggctctct ggggggacgg agctgcccct ggttttggga gccatcggcg 3360

gggctcagaa cagcctcgct gtggccgggt actgacctcc ccaccaccac tggcagccat 3420

ccctcccctt gccacccgtt ctgccttcca ggtgcagcat gtggtgtctc agaactgtga 3480

cgggctccac ctgaggagtg ggctgccgcg cacggccatc tccgagctcc acgggaacat 3540

gtacattgaa gtgagcagtc ctgcagggac ccagggtctc catgggcagg cgggtcccac 3600

tcactgtgcc ctcttgcctc taggtctgta cctcctgcgt tcccaacagg gagtacgtgc 3660

gggtgttcga tgtgacggag cgcactgccc tccacagaca ccagacaggc cggacctgcc 3720

acaagtgtgg gacccagctg cgggacacca ttgtgcactt tggggagagg gggacgttgg 3780

ggcagccttt gaactgggaa gcggcgaccg aggctgccag cagagcagac accatcctgt 3840

gtctagggtc cagcctgaag gtacgtgccg atgacacaat gagtgaaccg agcccctgcc 3900

cgcccgaggg tgtccagctc tgcggcccag cactgtacag acttgtccct tgtgtgtgtg 3960

cggtgtctgt ctgtctgctt ccacaggttc taaagaagta cccacgcctc tggtgcatga 4020

ccaagccccc tagccggcgg ccgaagcttt acatcgtgaa cctgcaggta actcgggtgc 4080

tgagagccac gtccttagat ctgggtctta gaacgcacag ccagagacac cccacaccca 4140

tgcaccaggg cggtctgata gggcccccgt gggtgctcag ggagcaccga ctgagcccgt 4200

aggggccaag gctgacaggc caccgggaag ggttgggctg ctgttactct cactcggctt 4260

tccctgtcct cagtggaccc cgaaggatga ctgggctgcc ctgaagctac atgggaagtg 4320

tgatgacgtc atgcggctcc tcatggccga gctgggcttg gagatccccg cctatagcag 4380

gtgagtgagc cgctgcagca gcctgcttcc ccgcacctct gtgtgctggg ccttgtctgt 4440

cttctctcgt gagctgagtg tggaggaagc tctgaggtgt ttgcagtggt gcctgaggca 4500

tgactgaagc gtggtggtct ccagagggcc tgacctcggt ggttggcgga gaccctgcgt 4560

gtgccactcc tgccctggct gatgtggcac acacaatccc cgcggggaga gggattctgc 4620

ccgcgtgctc ctgctccagg cctccccgtg gagctctccg agatgcctgg tgggaagcat 4680

ctggagggga cgagcactcg gcagctctgg tcagacagaa tctgtgtgct tggttttggg 4740

agttggcgta ctttgggaaa gcttaaacaa actgtgcctt aatacagaat ttgtgataat 4800

ttagacttgg tgtatgtatt gagtaaaaag tttacactct ctttctctgt gaattttcag 4860

ggtcttatag gggaaatcaa taacttcttt taatcaaagg gttcaagaaa ttaaggatcc 4920

cttcaccttc tgggcctggc acttcttgta tgttatgtgt gtggtgttct gtgatgtggg 4980

ctatcgtgta ctgtattttt tttttacatt aacttagctc attttcctta tcagtgcgta 5040

tctgtatctt aagttatgat ctgtggttct gcatctccgt cagacacatg ctttcttcac 5100

ggggtcgtct gtaggccacg cctccctagt cagctgggaa gggggagagg gtctggtcca 5160

cctgccccag cggtacaagt ggaaggtggg gcccagagtt gctagtgact catccctgga 5220

gacggaggca gccctggggc cactgctgcc ccaccctgtg tgtgcacgcc gctcagtggt 5280

ggacaaggac acggagtttt gaggagaccg agctagtgtg ggtgccgacc tttgagtcac 5340

cacctaagag gtgacctctc ccacatccgt tctgcagctt ggtaacaatg aagctgccgc 5400

caaccagagc cccgccgcag ttgacacggg agggaagggg atgggaaggc agggaccgca 5460

gacagctttc ccgagctggg gcaggtgtga ctgcgagagg ctcccaggcc cgcctgatgc 5520

cgctttccct ttttggcagg tggcaggatc ccattttctc actggcgact cccctgcgtg 5580

ctggtgaaga aggcagccac agtcggaagt cgctgtgcag aagcagagag gaggccccgc 5640

ctggggaccg gggtgcaccg cttagctcgg cccccatcct agggggctgg tttggcaggg 5700

gctgcacaaa acgcacaaaa aggaagaaag tgacgtaatc acgtgctcga tgaagaacag 5760

ttggcacttt gcagatggcc agtgtcacgg tgaaggctgg gttgccccca cgggtctagg 5820

gagaacgaac tctttgggga tgacattttc accgtgacat ttttagccat ttgtccttga 5880

ggaagcccct tgcactgctg cggttgtacc ctgatacggc ctggccatcg aggacacctg 5940

cccatccggc ctctgtgtca agaggtggca gccgcacctt tctgtgagaa cggaactcgg 6000

gttatttcag ccccggcctg cagagtggaa gcgcccagcg gcctttcctc gctcaccagg 6060

ccagtctcag ggcctcaccg tatttctact actacttaat gaaaaagtgt gaactttata 6120

gaatcctctc tgtactggat gtgcggcaga ggggtggctc cgagcctcgg ctctatgcag 6180

acctttttat ttctattaaa cgtttctgca ctggc 6215

<210>6

<211>1203

<212>DNA

<213>Homo sapiens

<400>6

atggcagccg ggggtctgag ccgctccgag cgcaaagcgg cggagcgggt ccggaggttg 60

cgggaggagc agcagaggga gcgcctccgc caggtgtcgc gcatcctgag gaaggcggcg 120

gcggagcgca gcgccgagga gggccggctg ctggccgaga gcgcggacct ggtaacggag 180

ctgcagggcc ggagccggcg gcgcgagggc ctgaagcggc ggcaggagga ggtgtgcgac 240

gacccggagg agctgcgggg gaaggtccgg gagctggcca gcgccgtccg gaacgccaaa 300

tacttggtcg tctacacagg cgcgggaatc agcacggcag cgtctatccc agactaccgg 360

ggccctaatg gagtgtggac actgcttcag aaagggagaa gcgttagtgc tgccgacctg 420

agcgaggccg agccaaccct cacccacatg agcatcaccc gtctgcatga gcagaagctg 480

gtgcagcatg tggtgtctca gaactgtgac gggctccacc tgaggagtgg gctgccgcgc 540

acggccatct ccgagctcca cgggaacatg tacattgaag tctgtacctc ctgcgttccc 600

aacagggagt acgtgcgggt gttcgatgtg acggagcgca ctgccctcca cagacaccag 660

acaggccgga cctgccacaa gtgtgggacc cagctgcggg acaccattgt gcactttggg 720

gagaggggga cgttggggca gcctttgaac tgggaagcgg cgaccgaggc tgccagcaga 780

gcagacacca tcctgtgtct agggtccagc ctgaaggttc taaagaagta cccacgcctc 840

tggtgcatga ccaagccccc tagccggcgg ccgaagcttt acatcgtgaa cctgcagtgg 900

accccgaagg atgactgggc tgccctgaag ctacatggga agtgtgatga cgtcatgcgg 960

ctcctcatgg ccgagctggg cttggagatc cccgcctata gcaggtggca ggatcccatt 1020

ttctcactgg cgactcccct gcgtgctggt gaagaaggca gccacagtcg gaagtcgctg 1080

tgcagaagca gagaggaggc cccgcctggg gaccggggtg caccgcttag ctcggccccc 1140

atcctagggg gctggtttgg caggggctgc acaaaacgca caaaaaggaa gaaagtgacg 1200

taa 1203

Claims

1. The application of lamivudine in preparing medicaments; the function of the medicine is as follows (a) and/or (b) and/or (c) and/or (d) and/or (e) and/or (f) and/or (g) and/or (h):

(a) delaying aging;

(b) delaying cell aging;

(c) delaying the aging of the mesenchymal stem cells;

2. The preparation method of the cell model comprises the following steps: reducing the content of SIRT7 protein in the stem cell and/or reducing the activity of SIRT7 protein in the stem cell and/or inhibiting the expression of coding gene of SIRT7 protein in the stem cell and/or editing the coding gene of SIRT7 protein in the stem cell to obtain the recombinant cell, wherein the recombinant cell or the passage cell thereof is a cell model.

3. The preparation method of the cell model comprises the following steps:

4. The preparation method of the cell model comprises the following steps:

5. The method of claim 2, 3 or 4, wherein: the stem cells are totipotent stem cells or pluripotent stem cells.

6. The method of claim 5, wherein: the pluripotent stem cells are embryonic stem cells or induced pluripotent stem cells.

7. The method of claim 3 or 4 or 5 or 6, wherein: the gene editing is realized through a CRISPR/Cas9 system.

8. A cell model produced by the method of any one of claims 3 to 7.

9. Any one of the following X1) -X4):

x1) for constructing the cell model of claim 8, consisting of a gRNA targeting the SIRT7 gene and a Cas9 nuclease;

x2) a system for constructing the cell model of claim 8, consisting of a vector expressing a gRNA targeting the SIRT7 gene and a vector expressing a Cas9 nuclease;

x3) for constructing the cell model of claim 8, consisting of a gRNA targeting exon 4 of the SIRT7 gene and a Cas9 nuclease;

x4) for constructing the cell model of claim 8, consisting of a vector expressing gRNA targeting exon 4 of SIRT7 gene and a vector expressing Cas9 nuclease.

10. The cell model of claim 8 applied to any one of the following Y1) -Y7):

y2) as a cell model for SIRT7 loss of function;

y3) as a cellular model for accelerated aging progression;

y4) for screening for substances that affect the expression of L INE1 in cells and/or affect the proliferative capacity of cells and/or affect the persistence capacity in cells and/or affect the senescence process of cells;