CN114958766B

CN114958766B - Construction method of aging cell model

Info

Publication number: CN114958766B
Application number: CN202210608729.1A
Authority: CN
Inventors: 张传茂; 王向阳
Original assignee: Peking University
Current assignee: Peking University
Priority date: 2022-05-31
Filing date: 2022-05-31
Publication date: 2023-12-26
Anticipated expiration: 2042-05-31
Also published as: CN114958766A

Abstract

The invention relates to the technical field of genetic engineering, in particular to a construction method of an aging cell model. The construction method comprises the following steps: in human skin fibroblasts, the protein progerin is overexpressed. The invention stably expresses protein progerin in human skin fibroblasts by a lentiviral vector transformation method, and constructs an aging cell model showing various aging indexes, which shows obvious phenomena such as DNA damage increase, chromatin abnormality, cell cycle retardation, cell nucleus abnormality, aging-related secretion phenotype increase and the like, and has important significance for scientific research and related drug research and development of presenility, aging-related diseases and other scientific problems.

Description

Construction method of aging cell model

Technical Field

The invention relates to the technical field of genetic engineering, in particular to a construction method of an aging cell model.

Background

There are various existing aging cell models including replicative aging cell models, oncogene-induced aging cell models, stress-type aging cell models, and the like. The replicative senescence cell model is mainly formed by gradually shortening telomeres after continuous division and proliferation of cells through high-frequency passage, and is typically characterized in that: reduced proliferation capacity of cells, cell cycle arrest, etc. Oncogene-induced senescence cell models, which are most typical in Ras expression induction, are characterized by unstable nuclei and gradual degradation. The stress type aging cell model is an aging cell model induced by external stimulus, such as oxidative stress, radiation irradiation, and the like. A typical feature of this model of aging cells is the presence of DNA damage, and the defect in DNA damage repair. The methods of construction of these senescent cell models are relatively complex and, due to the different methods of construction, there are large differences in the senescent phenotype exhibited. In addition, a single type of senescent cell model exhibits relatively few senescent phenotypes and cannot be used as a universal senescent cell model for scientific research and drug development.

The presenilin progerin is produced by alternative splicing following mutation of the gene LMNA, and it causes accelerated senescence, presenilin (Hutchinson-Gilford progeria syndrome), as well as expression during normal senescence. Typical conditions of presenility include a variety of aging-related diseases, such as: alopecia, subcutaneous fat loss, osteoporosis, atherosclerosis, chronic inflammation, tissue and organ fibrosis, myocardial infarction, apoplexy, etc. Thus, presenility is an advantageous tool in studying the mechanisms of aging and in developing anti-aging drugs. So far, the senescent cells of the presenility type are mainly derived from skin cells or blood cells of the presenility patient, and the cell sources are limited.

Due to the factors of small number of presenility patients, inconvenient tissue separation of patient samples, limited in-vitro culture of patient cells and the like, the sources of aging cells of the presenility type are limited, which severely restricts scientific research and drug development of the presenility and the aging-related diseases.

Disclosure of Invention

In order to solve the problems in the prior art, the invention provides a construction method of an aging cell model.

In a first aspect, the present invention provides a method for constructing a senescent cell model, comprising:

in human primary cells, the protein progerin is overexpressed.

Aiming at the problems of large difference, less aging phenotype and the like of the existing aging cell model, the invention constructs the aging cell model with various aging phenotypes by expressing presenilin progerin in human primary cells, and specifically comprises the following steps: increased DNA damage, cell cycle arrest, abnormal chromatin, abnormal nuclei, increased senescence-associated secretion phenotypes, and the like. The primary human cells required by the invention can be separated from the skin in a large quantity, and the materials are convenient to obtain and the cost is low.

Further, the human primary cells are human primary skin fibroblasts.

Further, the protein progerin comprises an amino acid sequence shown as SEQ ID NO. 1.

Further, the gene encoding the protein progerin was constructed into lentiviral vectors and then transfected into HK293T cells, and after collection of virus liquid, the human skin fibroblasts were infected.

Further, the gene encoding the protein progerin comprises a nucleotide sequence shown as SEQ ID NO. 2.

Further, the lentiviral vector comprises: pCDH, pQCXIP, pLVX or pLenti 6/TR.

Further, the method further comprises the following steps:

successfully impregnated human skin fibroblasts were screened using a flow cytometer.

The invention further provides an aging cell model constructed by the construction method.

The invention further provides application of the aging cell model in research of aging-related diseases.

Further, the aging-related disease includes one or more of presenility, arteriosclerosis, lipodystrophy, osteoporosis, alopecia, myocardial infarction, stroke, chronic inflammation, pulmonary fibrosis, liver fibrosis, heart fibrosis, kidney fibrosis, or spleen fibrosis.

The invention has the following beneficial effects:

the invention stably expresses protein progerin in human skin fibroblasts and constructs a GFP-progerin aging cell model. The aging cells constructed exhibit a variety of aging indicators including increased DNA damage, abnormal chromatin, cell cycle arrest, abnormal nuclei, increased aging-related secretory phenotypes, and the like. The invention provides a stable and uniform aging cell model, which has important significance for scientific research and related drug research and development of presenility, aging-related diseases and other scientific problems.

Drawings

FIG. 1 is a schematic diagram of the construction method of GFP-progerin senescent cells provided in example 1 of the present invention.

Fig. 2 is a schematic diagram showing the result of cellular immunofluorescence detection of DNA damage marker γh2ax provided in example 2 of the present invention.

FIG. 3 is a schematic diagram showing the result of the cellular immunofluorescence assay of heterochromatin binding protein HP 1. Alpha. Provided in example 2 of the invention.

FIG. 4 is a schematic diagram showing the result of the cellular immunofluorescence assay of heterochromatin marker H3K9me3 according to example 2 of the present invention.

FIG. 5 is a schematic diagram showing the result of the cellular immunofluorescence assay of centromere marker crest provided in example 2 of the present invention.

Fig. 6 is a schematic diagram showing the result of cellular immunofluorescence detection of nuclear pore complex marker nup153 according to example 2 of the present invention.

Fig. 7 is a schematic diagram of western blotting detection result of p16 protein in an presenility patient provided in example 2 of the invention.

FIG. 8 is a schematic diagram showing the Western blotting detection result of p16 in GFP-progerin senescent cells provided in example 2 of the present invention.

FIG. 9 is a schematic diagram showing the results of detection of senescence-associated secretory phenotype genes in a presenility patient according to example 2 of the invention.

FIG. 10 is a schematic diagram showing the results of detection of related secretory phenotype genes of GFP-progerin senescent cell model provided in example 2 of the present invention.

Detailed Description

The following examples are illustrative of the invention and are not intended to limit the scope of the invention.

Reagents and materials referred to in the following examples are commercially available unless otherwise indicated, and the methods of the experiments referred to are all routine in the art unless otherwise indicated.

Example 1

The embodiment provides a method for constructing an aging cell model, which specifically comprises the following steps:

1. experimental materials

And (3) cells: primary human skin fibroblasts (human dermal fibroblasts, HDF), HEK293T (ATCC).

And (3) a carrier: pCDH-CMV-MCS-EF1, psPAX2, pMD2.G, pEGFP-C1.

The medium used was as follows:

common medium: high sugar Dulbecco's Modified Eagle Medium (DMEM) +5-20% fetal bovine serum+100U/mL penicillin+100 μg/mL streptomycin;

virus packaging medium: high sugar Dulbecco's modified Eagle medium (DM EM) +20-40% fetal bovine serum+100U/mL penicillin+100 μg/mL streptomycin.

2. Construction flow

2.1 isolation of human skin fibroblasts

Primary human skin fibroblasts used in the present invention can be isolated by themselves, and commercial products can also be purchased.

The cell separation method is as follows:

first, skin tissue having a diameter of 1mm was removed from the skin of a volunteer, and rinsed 3 times with Phosphate Buffered Saline (PBS); then, removing redundant adipose tissues by using an ophthalmic scissors in an ultra clean workbench, cutting the rest tissues, and washing the rest tissues with PBS for 3 times; then adding 0.1-0.25% pancreatin to submerge all tissue pieces, and placing in a mixture containing 5% CO ₂ In a cell incubator of (C) for 5-10min at 37 ℃; after the digestion was completed, the digestion was terminated by adding DMEM medium containing 5-20% fetal bovine serum. Then, the cell mass was blown off by gently repeating the blowing and sucking with a pipette. Finally, the cell suspensions were transferred to 35mm dishes, 4mL of DMED medium containing 5-20% fetal bovine serum, 100U/mL penicillin, 100. Mu.g/mL streptomycin was added, and the mixture was placed in a cell incubator at 5% CO ₂ Culturing at 37deg.C for 4-6 days. As the culture proceeds, the medium may be appropriately supplemented according to the amount of the medium in the culture dish.

Finally, the isolated cells are primary human skin fibroblasts. In the whole cell separation process, aseptic operation is ensured, and double antibodies are added into the culture medium to avoid pollution. After the cells are separated, mycoplasma detection is carried out, and after no mycoplasma pollution is determined, the cells can be used for subsequent experiments. To facilitate subsequent experiments, as many HDF cells with low numbers of cryopreservation were performed as possible.

The skin sample of the donor is cut into small pieces by using the ophthalmology, digested by using pancreatin, and then placed in an incubator for culture, and the dissociated cells are skin fibroblasts. The isolated primary human skin fibroblasts (generation 3) were used in this example.

2.2 construction of vectors

(1) Cloning of the coding sequence of human progerin

Total RNA is extracted from blood or skin samples of the presenility patient, then cDNA library is obtained by RT-PCR, and finally the coding sequence is amplified by PCR using progerin primer.

(2) Construction of pCDH-GFP-progerin vector

The coding sequence of GFP was cloned from the pEGFP-C1 vector by PCR, and then the pCDH-CMV-MCS-EF1 vector was cut using double digestion with XbaI and NotI, and the coding sequences of GFP and progerin were inserted, respectively. Wherein GFP is located at the N-terminus of progerin. The vector was sequenced successfully and used in subsequent experiments.

The PCR primer sequences of progerin are as follows:

progerin F：5’-CAGTCTAGAATGGTGAGCAAGGGCGAGGA-3’

progerin R：5’-CAGGCGGCCGCTTACATGATGCTGCAGTTCT-3’。

2.3 viral packaging

HEK293T was passaged into 10cm dishes and 10mL of virus packaging medium was added and then placed in a medium containing 5% CO ₂ Is cultured in a cell culture vessel. After the cell density reached 40-70%, the constructed pCDH-GFP-progerin vector and psPAX2, pmd2.G were combined according to 2:1:1 were expressed by transfection using PEI. After 48 hours, cell culture supernatants were collected and pressed as supernatants: PEG-ittM virus sediment = 1:4, and the sediment was added and mixed and placed in a refrigerator at 4 ℃ for overnight sedimentation. The virus particles were then enriched by centrifugation at 1500g for 30min at 4℃and the virus was resuspended in 1mL PBS.

2.4 sorting senescent cells

HDF cells were passaged into 10cm dishes and cultured with 10mL of common medium. When the cell density reaches 40-70%Adding a proper amount of virus infection cells; after 6 hours, the fresh medium was changed. After the culture is continued for 24 to 48 hours, the culture medium in the culture dish is sucked off, and the culture dish is washed with PBS for 2 times, and each time is 2mL; then, the PBS is sucked off, 1mL of pancreatin with the concentration of 0.25% is added, and the culture dish is repeatedly shaken by both hands, so that the pancreatin uniformly infiltrates the bottom of the culture dish; finally, the mixture is placed in a mixture containing 5% CO ₂ In a cell incubator of (C) for 1-2min at 37 ℃; taking out the digested cells, adding 0.5mL of common culture medium into a culture dish, and stopping digestion; then repeatedly blowing and sucking by using a pipette, uniformly blowing cells, transferring the cells into a 1.5mL centrifuge tube, and centrifuging at 1000rpm/min for 4 minutes; the supernatant was aspirated, 1mL DMEM was added, gently and repeatedly aspirated, the cells were homogenized, GFP positive cells were sorted by flow sorter, and seeded for expansion. The cells obtained were GFP-progerin senescent cells.

As shown in FIG. 1, green fluorescence represents GFP-progerin, red fluorescence represents lamin labin B1, and blue fluorescence represents chromatin. The results show that the GFP-progerin aging cell is constructed by stably expressing GFP-progerin in normal skin fibroblasts through a lentiviral vector. GFP-progerin senescent cells exhibit abnormal states such as shrinkage of the nuclear membrane, budding of the nuclear membrane, and the like, relative to normal cells.

Example 2

This example is directed to GFP-progerin senescent cells obtained in example 1, and the relevant senescence indicators were detected by cellular immunofluorescence, western immunoblotting and RT-QPCR.

1. By skin fibroblast immunofluorescence experiment, gamma H2AX, HP1 alpha, H3K9me3, crest and nup153 indexes are detected, and the flow is as follows:

(1) GFP-progerin senescent cells were passaged into 35mm petri dishes with coverslips, 3ml of common medium was added and placed in a medium containing 5% CO ₂ Culturing at 37 ℃;

(2) When the cell density reached 70-100%, the coverslip was washed 3 times with PBS and then fixed with 4% Paraformaldehyde (PFA) for 20 minutes;

(3) Washing with PBS 3 times, and punching with 2.5% Triton-100 for 5min;

(4) After washing 3 times with PBS, the cells after fixation were labeled with antibodies. Antibodies to Progerin, γh2ax, HP 1a, H3K9me3, crest, nup153 were added to 3% bsa in PBS buffer at a ratio of 1:200, and the fixed cells were back-buckled in primary anti-dilution and incubated overnight at 4 ℃.

(5) The cells after incubation are dipped and washed gently in PBS buffer solution for three times;

(6) According to the corresponding species attribute of the primary antibody, the fluorescent secondary antibody is prepared according to the following formula 1:200 in a 3% BSA buffer, then the fixed cells were back-buckled in a secondary antibody diluent and incubated for 1hr at room temperature.

(7) Mowiol caplets, containing 1 μg/mL DNA dye DAPI and 2.5% of the anti-quencher DABCO, were heat-applied in an oven at 55deg.C in advance. After washing the cells after the secondary antibody incubation with PBS for 3 times, the cells were then back-buckled on thawed 15. Mu.L Mowiol caplets, dried at room temperature in the dark for 1 hour, and microscopic imaging was performed after the caplets were solidified.

(8) Microscopic imaging was performed using a DeltaVision microscope (equipped with an olympus IX-71 inverted microscope, 100×/1.4n.a. oil objective lens, and CCD camera); and the images were processed using the Volocity 6.1.1 software.

The final results of cellular immunofluorescence are shown in FIGS. 2-6, and are specifically as follows:

as shown in FIG. 2, green fluorescence represents progerin, red fluorescence represents DNA damage index γH2AX, and blue fluorescence represents chromatin. The results showed that in Normal skin fibroblasts (Normal cells), the DNA damage marker γh2ax was not detected; in skin fibroblasts (HGPS cells) of the presenility patient, the DNA damage marker γh2ax can be detected significantly; in the constructed GFP-progerin senescent cells (Normal cells+GFP-progerin), the expression of GFP-progerin also leads to an increase in the DNA damage marker γH2AX. These results indicate that the constructed GFP-progerin senescent cells exhibit increased DNA damage.

As shown in FIG. 3, green fluorescence represents progerin, red fluorescence represents heterochromatin binding protein HP 1. Alpha., and blue fluorescence represents chromatin. The results show that heterochromatin binding protein HP 1a can be detected in Normal skin fibroblasts (Normal cells); whereas in skin fibroblasts (HGPS cells) of the presenility patient, heterochromatin binding protein HP 1a is significantly reduced; in the constructed GFP-progerin senescent cells (Normal cells+GFP-progerin), the expression of GFP-progerin also leads to a reduction of the heterochromatin binding protein HP 1. Alpha.s. These results indicate that the constructed GFP-progerin senescent cells exhibit heterochromatin abnormalities.

As shown in FIG. 4, green fluorescence represents progerin, red fluorescence represents heterochromatin marker H3K9me3, and blue fluorescence represents chromatin. The results show that in Normal skin fibroblasts (Normal cells) the heterochromatin marker H3K9me3 can be detected; whereas in skin fibroblasts (HGPS cells) of the presenility patient, the heterochromatin marker H3K9me3 is significantly reduced; in the constructed GFP-progerin senescent cells (Normal cells+GFP-progerin), the expression of GFP-progerin also resulted in a decrease in the heterochromatin marker H3K9me 3. These results indicate that the constructed GFP-progerin senescent cells exhibit heterochromatin abnormalities.

As shown in FIG. 5, green fluorescence represents the presenilin progerin, red fluorescence represents the centromere marker crest, and blue fluorescence represents chromatin. The results show that in Normal skin fibroblasts (Normal cells), the centromere marker crest can be detected; whereas in skin fibroblasts (HGPS cells) of presenility patients, the centromere marker crest is significantly reduced; in constructed GFP-progerin senescent cells (Normal cells+GFP-progerin), GFP-progerin expression also resulted in a decrease in the centromere marker crest. These results indicate that the constructed GFP-progerin senescent cells exhibit abnormal centromeres.

As shown in FIG. 6, green fluorescence represents progerin, red fluorescence represents nuclear pore complex marker nup153, and blue fluorescence represents chromatin. The results showed that in Normal skin fibroblasts (Normal cells), the nuclear pore complex markers nup153 exhibited uniform distribution; whereas in skin fibroblasts (HGPS cells) of the presenility patient, the nuclear pore complex signature nup153 forms aggregates; in the constructed GFP-progerin senescent cells (Normal cells+GFP-progerin), GFP-progerin expression also resulted in the formation of aggregates from the nuclear pore complex markers nup 153. These results indicate that the constructed GFP-progerin senescent cells exhibit abnormal nuclear pore complex localization.

2. The invention further detects the expression of the senescence marker protein p16 by skin fibroblast western blotting, and the specific method is as follows:

(1) GFP-progerin senescent cells were passaged into 35mm dishes, 3ml of common medium was added and placed in a medium containing 5% CO ₂ Culturing at 37 ℃;

(2) When the cell density reaches 80-100%, sucking off the culture medium in the culture dish, washing 2 times with PBS (phosphate buffer solution) and 2mL each time; then, the PBS is sucked off, 1mL of pancreatin with the concentration of 0.25% is added, and the culture dish is repeatedly shaken by both hands, so that the pancreatin uniformly infiltrates the bottom of the culture dish; finally, the mixture is placed in a mixture containing 5% CO ₂ In a cell incubator of (C) for 1-2min at 37 ℃;

(3) Taking out the digested cells, adding 0.5mL of common culture medium into a culture dish, and stopping digestion; then repeatedly blowing and sucking by using a pipette, uniformly blowing cells, transferring the cells into a 1.5mL centrifuge tube, centrifuging at 1000rpm/min for 4 minutes, and enriching the cells;

(4) Sucking the supernatant, re-suspending the cells with 20-100 μl of PBS, adding equal volume of sample buffer, mixing, and decocting in 95 deg.C vibrator for 10min;

(5) Performing immunoblotting analysis experiment on the obtained sample, preparing 5-12% SDS-PAGE gel, and performing electrophoresis separation on the protein under the condition of 120V constant pressure;

(6) Taking out SDS-PAGE gel after electrophoresis, removing upper concentrated gel, placing in a transfer membrane buffer solution (25mM Tris,192mM glycine and 20% methanol), stacking according to the sequence of sponge, filter paper, nitrocellulose membrane, albumin gel, filter paper and sponge, removing bubbles, clamping by a transfer membrane clamp, and performing wet transfer in the transfer membrane solution for 2 hours under the constant current condition of 300 mA;

(7) After the transfer, the nitrocellulose membrane was taken out and placed in Fast Green dye to preliminarily observe the transfer effect, and then the dye was washed off with TTBS (20 mM Tris-Cl, pH 7.4, 500mM NaCl,0.3%Tween-20). Sealing with 3% skimmed milk diluted in TTBS at room temperature for 30-60min;

(8) After blocking, primary antibodies were labeled overnight at 4deg.C with antibodies to p16, β -actin, GFP (primary antibodies were diluted in TTBS with 3% skim milk, typically at a ratio of 1:500-1:5000);

(9) The next day, the nitrocellulose membrane was washed three times with TTBS for 10min each time; then incubating for 30-60min at room temperature with HRP-conjugated secondary antibody (the secondary antibody is diluted in TTBS containing 3% skimmed milk at a ratio of typically 1:5000-1:20000);

(10) Washing the nitrocellulose membrane with TTBS three times for 10min each time;

(11) Finally, the nitrocellulose membrane coupled with the secondary antibody is reacted with a color development solution, and is exposed and developed in a darkroom by using an X-film negative.

The results are shown in FIG. 7, where β -actin represents an internal control and p16 represents a senescence marker protein. The results showed that the expression of senescence-associated protein p16 was increased in skin fibroblast samples (HGPS) from presenility patients relative to Normal skin fibroblast samples (Normal).

As shown in FIG. 8, β -actin represents an internal control and p16 represents an aging marker protein. The results show that in the constructed GFP-progerin senescent cells, the expression of GFP-progerin results in increased expression of the senescence marker protein p 16. These results indicate that the constructed GFP-progerin senescent cells showed an increase in the senescence marker protein p16, and a cell cycle arrest.

3. The invention further detects the expression of marker genes of five senescence-associated secretion phenotypes of IL1A, IL1B, IL6, IL8 and MMP3 through an RT-QPCR (quantitative PCR) experiment, and the method comprises the following steps:

(1) GFP-progerin senescent cells were passaged into 35mm dishes, 3mL of common medium was added, and placed in a medium containing 5% CO ₂ Culturing at 37 ℃;

(2) When the cell density reaches 80-100%, sucking off the culture medium in the culture dish, washing 2 times with PBS (phosphate buffer solution) and 2mL each time; the PBS was then aspirated off and 1mL Trizol was added. The cells were then thoroughly lysed in Trizol by repeated pipetting with a pipette and transferred to a 1.5mL centrifuge tube.

(3) Extracting total RNA according to the operation instruction of Trizol;

(4) Reverse transcription was performed using total RNA, followed by fluorescent quantitative PCR to detect the expression levels of five marker genes IL1A, IL1B, IL6, IL8, MMP 3.

The PCR primer sequences used were as follows:

IL1A-F:5’-TGTAAGCTATGGCCCACTCCA-3’，

IL1A-R:5’-AGAGACACAGATTGATCCATGCA-3’；

IL1B-F:5’-CTCTCTCCTTTCAGGGCCAA-3’，

IL1B-R:5’-GAGAGGCCTGGCTCAACAAA-3’；

IL6-F:5’-CACCGGGAACGAAAGAGAAG-3’，

IL6-R:5’-TCATAGCTGGGCTCCTGGAG-3’；

IL8-F:5’-ACATGACTTCCAAGCTGGCC-3’，

IL8-R:5’-CAGAAATCAGGAAGGCTGCC-3’；

MMP3-F:5’-GGATGCCAGGAAAGGTTCTG-3’，

MMP3-R:5’-CCAGGTGTGGAGTTCCTGATGT-3’。

the results are shown in FIG. 9, where the expression of five senescence-associated secretory phenotype genes, IL1A, IL1B, IL6, IL8, MMP3, was shown to be significantly increased in skin fibroblast samples (HGPS) from presenility patients relative to Normal skin fibroblast samples (Normal).

As shown in FIG. 10, in the constructed GFP-progerin senescent cells, GFP-progerin expression resulted in the expression of five senescence-associated secretory phenotype genes, IL1A, IL1B, IL6, IL8, MMP3, being shown to be significantly increased. These results indicate that the constructed GFP-progerin senescent cells exhibit an increase in the senescence-associated secretory phenotype.

While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.

Sequence listing

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cgtgcttcct ctcactcatc ccagacacag ggtgggggca gcgtcaccaa aaagcgcaaa 1260

ctggagtcca ctgagagccg cagcagcttc tcacagcacg cacgcactag cgggcgcgtg 1320

gccgtggagg aggtggatga ggagggcaag tttgtccggc tgcgcaacaa gtccaatgag 1380

gaccagtcca tgggcaattg gcagatcaag cgccagaatg gagatgatcc cttgctgact 1440

taccggttcc caccaaagtt caccctgaag gctgggcagg tggtgacgat ctgggctgca 1500

ggagctgggg ccacccacag cccccctacc gacctggtgt ggaaggcaca gaacacctgg 1560

ggctgcggga acagcctgcg tacggctctc atcaactcca ctggggaaga agtggccatg 1620

cgcaagctgg tgcgctcagt gactgtggtt gaggacgacg aggatgagga tggagatgac 1680

ctgctccatc accaccacgg ctcccactgc agcagctcgg gggaccccgc tgagtacaac 1740

ctgcgctcgc gcaccgtgct gtgcgggacc tgcgggcagc ctgccgacaa ggcatctgcc 1800

agcggctcag gagcccagag cccccagaac tgcagcatca tgtaa 1845

<210> 3

<211> 29

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 3

cagtctagaa tggtgagcaa gggcgagga 29

<210> 4

<211> 31

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 4

caggcggccg cttacatgat gctgcagttc t 31

<210> 5

<211> 21

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 5

tgtaagctat ggcccactcc a 21

<210> 6

<211> 23

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 6

agagacacag attgatccat gca 23

<210> 7

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 7

ctctctcctt tcagggccaa 20

<210> 8

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 8

gagaggcctg gctcaacaaa 20

<210> 9

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 9

caccgggaac gaaagagaag 20

<210> 10

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 10

tcatagctgg gctcctggag 20

<210> 11

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 11

acatgacttc caagctggcc 20

<210> 12

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 12

cagaaatcag gaaggctgcc 20

<210> 13

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 13

ggatgccagg aaaggttctg 20

<210> 14

<211> 22

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 14

ccaggtgtgg agttcctgat gt 22

Claims

1. A method of constructing a model of senescent cells, comprising:

in human primary cells, the protein progerin is overexpressed;

the amino acid sequence of the protein progerin is shown as SEQ ID NO. 1.

2. The method of claim 1, wherein the human primary cells are human primary skin fibroblasts.

3. The construction method according to claim 2, comprising:

the gene encoding the protein progerin is constructed into a lentiviral vector, then HK293T cells are transfected, and after virus liquid is collected, the human skin fibroblasts are infected.

4. The method according to claim 3, wherein the nucleotide sequence encoding the protein progerin is shown in SEQ ID NO. 2.

5. The method of claim 3, wherein the lentiviral vector comprises: pCDH, pQCXIP, pLVX or pLenti 6/TR.

6. A method of construction according to claim 3, further comprising:

7. The senescent cell model constructed by the construction method according to any one of claims 1 to 6.

8. Use of the senescent cell model according to claim 7 in the study of a senescence-associated disease; the study was aimed at non-disease diagnosis and treatment.