A method for screening anti-aging drugs

Abstract

The present application relates to the technical field of genetic engineering, and particularly relates to a screening method of an anti-aging drug. The screening method comprises: treating cells overexpressing GFP-progerin protein with a drug to be screened, and comparing the cells overexpressing GFP-progerin protein without treatment of the drug to be screened; and judging the anti-aging effect of the drug to be screened according to the change of cell morphology. The present application finds that progerin protein positioned on the nuclear membrane can make cells show obvious aging characteristics, including nuclear membrane shrinkage and nuclear membrane budding. Progerin protein positioned in the nucleus does not make cells show obvious aging characteristics. When a drug has an anti-aging effect, progerin protein positioned on the nuclear membrane will be transferred to the nucleus. The present application provides a screening method of an anti-aging drug based on the above, which can accurately and efficiently screen an anti-aging drug.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of genetic engineering, and particularly relates to a screening method of an anti-aging drug. BACKGROUND

[0002] The senescent cells exhibit a variety of typical characteristics, including increased DNA damage, increased p16, increased beta-galactosidase activity, increased senescence-associated secretory phenotype, etc. The traditional anti-aging drug development idea is to kill the senescent cells, and the specific strategy is to develop drugs according to the typical characteristics of the senescent cells. For example, specifically removing the cells with high expression of p16, designing a lead drug and killing cells by using the activity of beta-galactosidase, inhibiting the expression of senescence-associated secretory factors, etc. The anti-aging drugs screened by these methods can inhibit cell aging and adult aging to a certain extent.

[0003] The typical characteristics of the senescent cells also appear in normal cells, for example, the senescence-associated secretory factors can promote embryonic structure recognition, tissue remodeling and repair, etc., and the inhibition of the expression thereof will disturb the normal functions of the body. The beta-galactosidase is also expressed in non-senescent tissue cells, such as bone marrow. Therefore, the anti-aging drug screening based on the characteristics of the senescent cells has a large side effect, which affects the functions of the normal tissue cells. SUMMARY

[0004] In order to solve the problems in the prior art, the present application provides a screening method of an anti-aging drug, which can accurately and quickly screen the drugs with anti-aging effect based on the principle that the localization of progerin protein on the nuclear membrane in the cells overexpressing GFP-progerin protein will change based on the anti-aging effect of the drugs.

[0005] In a first aspect, the present application provides a screening method of an anti-aging drug, comprising:

[0006] The cells overexpressing GFP-progerin protein are treated by a drug to be screened, and the cells overexpressing GFP-progerin protein without the treatment of the drug to be screened are compared, and the anti-aging effect of the drug to be screened is determined according to the change of the cell morphology.

[0007] Further, the cells overexpressing GFP-progerin protein are primary cells overexpressing GFP-progerin protein.

[0008] Further, the progerin protein comprises an amino acid sequence as shown in SEQ ID NO. 1.

[0009] Further, the cell overexpressing the GFP-progerin protein is prepared by the following method: a gene encoding the progerin protein is constructed into a lentivirus vector, then the HK293T cell is transfected, and the virus liquid is collected to infect the primary cell.

[0010] Further, the gene encoding the progerin protein comprises the nucleotide sequence shown in SEQ ID NO. 2.

[0011] Further, the primary cell is a human primary skin fibroblast cell.

[0012] Further, the lentivirus vector comprises one or more of pCDH, pQCXIP, pLVX or pLenti6 / TR.

[0013] Further, the anti-aging effect of the drug to be screened is determined according to the change in the cell morphology.

[0014] When one or more of the nuclear membrane budding, nuclear membrane shrinkage, heterochromatin abnormality or micronucleus of the cell overexpressing the GFP-progerin protein treated by the drug to be screened is inhibited, it is determined that the drug to be screened has an anti-aging effect.

[0015] Further, the anti-aging effect of the drug to be screened is determined according to the change in the cell morphology.

[0016] When the localization of the progerin protein in the cell overexpressing the GFP-progerin protein treated by the drug to be screened changes, it is determined that the drug to be screened has an anti-aging effect.

[0017] Further, the change in the localization is that the localization on the nuclear membrane changes to the localization in the nucleus.

[0018] The present application has the following beneficial effects:

[0019] The present application provides an anti-aging drug screening method based on the GFP-progerin aging cell model, which can realize high-throughput and high-precision anti-aging drug screening. The present application provides an efficient, stable and precise drug screening platform, which can realize the screening of a maximum of 1536 small molecule compounds at a time, and achieve high-throughput and high-precision drug screening. With the aid of this screening method, the present application successfully screens a plurality of candidate drugs with anti-aging function, and it is verified that the candidate drugs also have the effect of inhibiting aging for patient cells, which provides a new possibility for the treatment of progeria, aging, vascular sclerosis, tissue and organ fibrosis, aging inflammation and other diseases. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 Figure 1 shows the results of cell immunofluorescence detection of abnormal nuclei caused by GFP-progerin provided in Example 1 of the present application.

[0021] Figure 2 Figure 2 shows the results of statistics of the proportion of nuclear membrane budding caused by GFP-progerin provided in Example 1 of the present application.

[0022] Figure 3 Figure 3 shows the effect of the localization of progerin protein in the GFP-progerin senescent cell model provided in Example 1 of the present application on cells.

[0023] Figure 4 Figure 4 shows the overall flowchart of anti-aging drug screening provided in Example 1 of the present application.

[0024] Figure 5 Figure 5 shows the flowchart of high-throughput screening of anti-aging drugs provided in Example 1 of the present application.

[0025] Figure 6 Figure 6 shows the effect of candidate drug 1 on the localization of progerin provided in Example 2 of the present application.

[0026] Figure 7 Figure 7 shows the effect of candidate drugs 2 and 3 on the localization of progerin provided in Example 2 of the present application.

[0027] Figure 8 Figure 8 shows the effect of candidate drugs 1, 2 and 3 on nuclear membrane budding provided in Example 2 of the present application.

[0028] Figure 9 Figure 9 shows the effect of candidate drugs 1, 2 and 3 on the localization of progerin in the cells of progeria patients provided in Example 2 of the present application.

[0029] Figure 10 Figure 10 shows the effect of candidate drugs 1, 2 and 3 on nuclear membrane budding in the cells of progeria patients provided in Example 2 of the present application. DETAILED DESCRIPTION

[0030] The following examples are used to illustrate the present application, but are not used to limit the scope of the present application.

[0031] Example 1

[0032] This example provides a screening method for anti-aging drugs, and the flowchart is as follows:

[0033] 1. Experimental materials

[0034] Senescent cell model: GFP-progerin senescent cell model;

[0035] Culture medium: high glucose Dulbecco's modified Eagle medium (DMEM) + 5-20% fetal bovine serum + 100 U / mL penicillin + 100 μg / mL streptomycin;

[0036] The GFP-progerin senescent cell model is prepared by the following method:

[0037] (1) Isolation of human skin fibroblasts

[0038] First, a 1 mm diameter skin tissue is taken from the skin of a volunteer, and washed with phosphate buffer solution (PBS) for 3 times; then, the excess adipose tissue is removed and the remaining tissue is cut into pieces with an ophthalmic scissors in a clean bench, and washed with PBS for 3 times; then, 0.1-0.25% trypsin is added to immerse all the tissue pieces, and placed in a cell culture box containing 5% CO2, and digested at 37°C for 5-10 min; after the digestion is completed, the digestion is terminated by adding DMEM medium containing 5-20% fetal bovine serum. Then, the cell clusters are blown apart by repeatedly blowing and sucking with a pipette. Finally, the cell suspension is transferred into 35 mm culture dishes, and 4 mL of DMED medium containing 5-20% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin is added, and placed in a cell culture box under the condition of 5% CO2 and 37°C for 4-6 days. During the culture, the medium can be appropriately supplemented according to the amount of medium in the culture dish.

[0039] Finally, the isolated cells are primary human skin fibroblasts. During the whole cell isolation process, sterile operation is ensured, and double antibiotics are added to the culture medium to avoid contamination. After the cells are isolated, mycoplasma detection is performed to determine whether there is mycoplasma contamination, and only after it is determined that there is no mycoplasma contamination can the cells be used for subsequent experiments. In order to facilitate subsequent experiments, as many HDF cells as possible with low passage number are cryopreserved.

[0040] The skin sample of the donor is cut into small pieces with an ophthalmic scissors, and after trypsin digestion, the cells are placed in a culture box for culture, and the released cells are skin fibroblasts. The primary human skin fibroblasts (passage 3) used in this example are used.

[0041] (2) Construction of vector

[0042] Cloning of human progerin coding sequence:

[0043] Total RNA is extracted from the blood or skin sample of a progeria patient, and then a cDNA library is obtained by RT-PCR, and finally the coding sequence is amplified by PCR using primers of progerin.

[0044] Construction of pCDH-GFP-progerin vector:

[0045] The coding sequence of GFP was cloned from pEGFP-C1 vector by PCR, and then the pCDH-CMV-MCS-EF1 vector was cut by Xba I and Not I double enzyme, and the coding sequences of GFP and progerin were inserted respectively. Among them, GFP is located at the N-terminal of progerin. After successful vector sequencing, it was used for subsequent experiments.

[0046] The PCR primer sequence of progerin is as follows:

[0047] progerin F: 5'-CAGTCTAGAATGGTGAGCAAGGGCGAGGA-3'

[0048] progerin R: 5'-CAGGCGGCCGCTTACATGATGCTGCAGTTCT-3'.

[0049] (3) Virus packaging

[0050] HEK293T was passaged into a 10 cm culture dish, and 10 mL of virus packaging medium was added, and then placed in a cell culture incubator containing 5% CO2 for culture. After the cell density reached 40-70%, the constructed pCDH-GFP-progerin vector and psPAX2, pMD2.G were transfected and expressed according to the ratio of 2:1:1 using PEI. After 48 hours, the cell culture supernatant was collected, and the sedimentation solution was added according to the ratio of supernatant: PEG-itTM virus sedimentation solution = 1:4, and then mixed and placed in a 4°C refrigerator overnight for sedimentation. Then 4°C, 1500g centrifugation for 30min, enrichment of virus particles, and resuspension of virus with 1mL PBS.

[0051] (4) Sorting of senescent cells

[0052] Subculture the HDF cells into 10 cm culture dish and add 10 mL normal medium for culture. When the cell density reaches 40-70%, add appropriate amount of virus to infect the cells; 6 hours later, replace the fresh medium. Continue to culture for 24-48 hours, then aspirate the medium in the culture dish, wash twice with PBS, 2 mL each time; then aspirate the PBS, add 1 mL trypsin with a concentration of 0.25%, shake the culture dish repeatedly with both hands to make the trypsin evenly infiltrate the bottom of the culture dish; finally, place it in a cell culture incubator containing 5% CO2, digest for 1-2 min at 37°C; take out the digested cells, add 0.5 mL normal medium to the culture dish to terminate the digestion; then blow and suck repeatedly with a pipette to evenly blow the cells, and transfer them to a 1.5 mL centrifuge tube, centrifuge at 1000 rpm / min for 4 min; aspirate the supernatant, add 1 mL DMEM, gently blow and suck repeatedly to mix the cells, sort out the GFP-positive cells through a flow cytometer, and inoculate and expand. The obtained cells are the GFP-progerin senescent cells.

[0053] 2. Drug screening process

[0054] The screening process is shown in Figure 4 , and is as follows:

[0055] (1) Inoculate cells

[0056] Subculture the GFP-progerin senescent cells into 10 cm culture dishes and add 10 mL medium for culture. When the cell density reaches 90-100%, digest the senescent cells with 0.25% trypsin, blow the cells evenly, and inoculate them into 96-well plates for culture.

[0057] (2) Drug treatment

[0058] When the cell density reaches 60-80%, add small molecule compounds to each well of the 96-well plate, and treat for 24 hours.

[0059] (3) High-throughput screening

[0060] Place the treated GFP-progerin senescent cells on a high-content fluorescence microscope for observation, and capture the fluorescence image of a large field of view. As shown in Figure 5 , inoculate the GFP-progerin senescent cells into 96-well plates, treat them with small molecule compounds for 24 hours, then screen them through a high-content fluorescence microscope, and the screening standard is that the GFP-progerin is located in the nucleus. Finally, screen the single well of the 96-well plate in which the GFP-progerin has changed its location, and the corresponding small molecule compound is the candidate drug.

[0061] (4) Patient cell verification

[0062] The skin fibroblasts of the progeria patient are treated with the candidate drug to further verify the effectiveness of the candidate drug. The small molecule compound effective on both the GFP-progerin aging cells and the progeria patient cells is the final candidate drug screened.

[0063] As shown in Figure 1 , the localization of GFP-progerin-WT on the nuclear membrane causes nuclear membrane budding, nuclear membrane shrinkage (WT), while the mutant GFP-progerin-C661S is localized in the nucleus and does not cause nuclear abnormalities (C661S).

[0064] As shown in Figure 2 , compared with GFP-progerin, the mutant GFP-C661S causes a lower proportion of nuclear membrane budding. These results show that the localization of progerin in the nucleus can improve nuclear membrane abnormalities.

[0065] As shown in Figure 3 , the localization of GFP-progerin on the nuclear membrane causes nuclear membrane budding, nuclear membrane shrinkage (left), while inhibiting the localization of GFP-progerin on the nuclear membrane restores the cell to normal (right).

[0066] As shown in Figure 4 , the process of anti-aging drug screening includes: inoculation of GFP-progerin aging cells, drug treatment, high-content fluorescence microscope screening, and verification of progeria patient cells.

[0067] Example 2

[0068] This example verifies the anti-aging drug screened in Example 1 through a cell immunofluorescence experiment.

[0069] 1. The experimental method of cell immunofluorescence is as follows:

[0070] (1) The cells are subcultured into 35mm culture dishes coated with coverslips, 3ml of culture medium is added, and the culture is placed in a cell culture incubator containing 5% CO2, and cultured at 37°C;

[0071] (2) When the cell density reaches 70-100%, the coverslips are washed with PBS for 3 times, and then fixed with 4% paraformaldehyde (PFA) for 20 minutes;

[0072] (3) After washing with PBS for 3 times, punch with 2.5% Triton-100 for 5min;

[0073] (4) After washing three times with PBS, the fixed cells were labeled with antibodies. Antibodies for Progerin, emerin, lamin A / C, and lamin B1 were added to 3% BSA in PBS buffer at a ratio of 1:200. The fixed cells were then inverted in the primary antibody dilution solution and incubated overnight at 4°C.

[0074] (5) After incubation, gently wash the cells in PBS buffer three times.

[0075] (6) Based on the species attribute corresponding to the primary antibody, the fluorescent secondary antibody was diluted in 3% BSA buffer at a ratio of 1:200. Then, the fixed cells were inverted in the secondary antibody dilution solution and incubated at room temperature for 1 hour.

[0076] (7) Preheat the Mowiol mounting medium, which contains 1 μg / mL DNA dye DAPI and 2.5% anti-quenching agent DABCO, in a 55°C oven. After the cells have been incubated with the secondary antibody, wash them three times with PBS, then invert them onto the melted 15 μL Mowiol mounting medium and dry them at room temperature in the dark for 1 hour. After the mounting medium has solidified, perform microscopic imaging.

[0077] (8) Microscopic imaging was performed using a DeltaVision microscope (equipped with an Olympus IX-71 inverted microscope, a 100× / 1.4NA oil objective and a CCD camera); and the images were processed using Volocity 6.1.1 software.

[0078] 2. Experimental Results

[0079] like Figure 6 As shown, green fluorescence represents the early aging protein GFP-progerin, red fluorescence represents the nuclear membrane protein emerin, purple fluorescence represents the lamin B1 protein, and blue fluorescence represents chromatin. The results indicate that the screened candidate drug 1 can alter the localization of GFP-progerin, causing it to aggregate within the cell nucleus.

[0080] like Figure 7 As shown, green fluorescence represents the progerin GFP-progerin, red fluorescence represents lamin A / C, purple fluorescence represents lamin B1, and blue fluorescence represents chromatin. The results indicate that the screened candidate drugs 2 and 3 can alter the localization of GFP-progerin, causing it to aggregate in the cell nucleus.

[0081] like Figure 8As shown, compared with the control group, the nuclear membrane budding rate was significantly reduced in the treatment groups of candidate drugs 1, 2, and 3. These results indicate that candidate drugs 1, 2, and 3 can significantly inhibit nuclear membrane budding.

[0082] like Figure 9 As shown, green fluorescence represents progerin, red fluorescence represents lamin B1, and blue fluorescence represents chromatin. The results showed that the screened candidate drugs 1, 2, and 3 could alter the localization of progerin in the cells of progeria patients, causing it to aggregate in the nucleus.

[0083] like Figure 10 As shown, compared with the control group, the nuclear membrane budding rate was significantly reduced in the treatment groups of candidate drugs 1, 2, and 3. These results indicate that candidate drugs 1, 2, and 3 can significantly inhibit nuclear membrane budding in cells from progeria patients.

[0084] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention. sequence list <110> Beijing University <120> A screening method for anti-aging drugs <130> KHP221115094.5 <160> 4 <170> SIPOSequenceListing 1.0 <210> 1 <211> 614 <212> PRT <213> Artificial Sequence <400> 1 Met Glu Thr Pro Ser Gln Arg Arg Ala Thr Arg Ser Gly Ala Gln Ala 1 5 10 15 Ser Ser Thr Pro Leu Ser Pro Thr Arg lie Thr Arg Leu Gin Glu Lys 20 25 30 Glu Asp Leu Gin Glu Leu Asn Asp Arg Leu Ala Val Tyr lie Asp Arg 35 40 45 Val Arg Ser Leu Glu Thr Glu Asn Ala Gly Leu Arg Leu Arg lie Thr 50 55 60 Glu Ser Glu Glu Val Val Ser Arg Glu Val Ser Gly lie Lys Ala Ala 65 70 75 80 Tyr Glu Ala Glu Leu Gly Asp Ala Arg Lys Thr Leu Asp Ser Val Ala 85 90 95 Lys Glu Arg Ala Arg Leu Gin Leu Glu Leu Ser Lys Val Arg Glu Glu 100 105 110 Phe Lys Glu Leu Lys Ala Arg Asn Thr Lys Lys Glu Gly Asp Leu lie 115 120 125 Ala Ala Gin Ala Arg Leu Lys Asp Leu Glu Ala Leu Leu Asn Ser Lys 130 135 140 Glu Ala Ala Leu Ser Thr Ala Leu Ser Glu Lys Arg Thr Leu Glu Gly 145 150 155 160 Glu Leu His Asp Leu Arg Gly Gin Val Ala Lys Leu Glu Ala Ala Leu 165 170 175 Gly Glu Ala Lys Lys Gln Leu Gln Asp Glu Met Leu Arg Arg Val Asp 180 185 190 Ala Glu Asn Arg Leu Gln Thr Met Lys Glu Glu Leu Asp Phe Gln Lys 195 200 205 Asn Ile Tyr Ser Glu Glu Leu Arg Glu Thr Lys Arg Arg His Glu Thr 210 215 220 Arg Leu Val Glu Ile Asp Asn Gly Lys Gln Arg Glu Phe Glu Ser Arg 225 230 235 240 Leu Ala Asp Ala Leu Gln Glu Leu Arg Ala Gln His Glu Asp Gln Val 245 250 255 Glu Gln Tyr Lys Lys Glu Leu Glu Lys Thr Tyr Ser Ala Lys Leu Asp 260 265 270 Asn Ala Arg Gln Ser Ala Glu Arg Asn Ser Asn Leu Val Gly Ala Ala 275 280 285 His Glu Glu Leu Gln Gln Ser Arg Ile Arg Ile Asp Ser Leu Ser Ala 290 295 300 Gln Leu Ser Gln Leu Gln Lys Gln Leu Ala Ala Lys Glu Ala Lys Leu 305 310 315 320 Arg Asp Leu Glu Asp Ser Leu Ala Arg Glu Arg Asp Thr Ser Arg Arg 325 330 335 Leu Leu Ala Glu Lys Glu Arg Glu Met Ala Glu Met Arg Ala Arg Met 340 345 350 Gln Gln Gln Leu Asp Glu Tyr Gln Glu Leu Leu Asp Ile Lys Leu Ala 355 360 365 Leu Asp Met Glu Ile His Ala Tyr Arg Lys Leu Leu Glu Gly Glu Glu 370 375 380 Glu Arg Leu Arg Leu Ser Pro Ser Pro Thr Ser Gln Arg Ser Arg Gly 385 390 395 400 Arg Ala Ser Ser His Ser Ser Gln Thr Gln Gly Gly Gly Ser Val Thr 405 410 415 Lys Lys Arg Lys Leu Glu Ser Thr Glu Ser Arg Ser Ser Phe Ser Gln 420 425 430 His Ala Arg Thr Ser Gly Arg Val Ala Val Glu Glu Val Asp Glu Glu 435 440 445 Gly Lys Phe Val Arg Leu Arg Asn Lys Ser Asn Glu Asp Gln Ser Met 450 455 460 Gly Asn Trp Gln Ile Lys Arg Gln Asn Gly Asp Asp Pro Leu Leu Thr 465 470 475 480 Tyr Arg Phe Pro Pro Lys Phe Thr Leu Lys Ala Gly Gln Val Val Thr 485 490 495 Ile Trp Ala Ala Gly Ala Gly Ala Thr His Ser Pro Pro Thr Asp Leu 500 505 510 Val Trp Lys Ala Gln Asn Thr Trp Gly Cys Gly Asn Ser Leu Arg Thr 515 520 525 Ala Leu Ile Asn Ser Thr Gly Glu Glu Val Ala Met Arg Lys Leu Val 530 535 540 Arg Ser Val Thr Val Val Glu Asp Asp Glu Asp Glu Asp Gly Asp Asp 545 550 555 560 Leu Leu His His His His Gly Ser His Cys Ser Ser Ser Gly Asp Pro 565 570 575 Ala Glu Tyr Asn Leu Arg Ser Arg Thr Val Leu Cys Gly Thr Cys Gly 580 585 590 Gln Pro Ala Asp Lys Ala Ser Ala Ser Gly Ser Gly Ala Gln Ser Pro 595 600 605 Gln Asn Cys Ser Ile Met 610 <210> 2 <211> 1845 <212> DNA <213> Artificial Sequence <400> 2 atggagaccc cgtcccagcg gcgcgccacc cgcagcgggg cgcaggccag ctccactccg 60 120. ctgtcgccca cccgcatcac ccggctgcag gagaaggagg acctgcagga gctcaatgat cgcttggcgg tctacatcga ccgtgtgcgc tcgctggaaa cggagaacgc agggctgcgc cttcgcatca ccgagtctga agaggtggtc agccgcgagg tgtccggcat caaggccgcc 240 tacgaggccg agctcgggga tgcccgcaag acccttgact cagtagccaa ggagcgcgcc cgcctgcagc tggagctgag caaagtgcgt gaggagttta aggagctgaa agcgcgcaat 360 accaagaagg agggtgacct gatagctgct caggctcggc tgaaggacct ggaggctctg ctgaactcca aggaggccgc actgagcact gctctcagtg agaagcgcac gctggagggc 480 gagctgcatg atctgcgggg ccaggtggcc aagcttgagg cagccctagg tgaggccaag 540 aagcaacttc aggatgat gctgcggcgg gtggatgctg agaacaggct gcagaccatg aaggaggac tggacttcca gaagacatc tacagtgagg agctgcgtga gaccaagcgc cgtcatgaga cccgactggt ggagattgac aatgggaagc agcgtgagtt tgagagccgg 720 ctggcggatg cgctgcagga actgcgggcc cagcatgagg accaggtgga gcagtataag 780 aaggagctgg agaagactta ttctgccaag ctggacaatg ccaggcagtc tgctgagagg 840 aacagcaacc tggtgggggc tgcccacgag gagctgcagc agtcgcgcat ccgcatcgac 900 agcctctctg cccagctcag ccagctccag aagcagctgg cagccaagga ggcgaagctt 960 cgagacctgg aggactcact ggcccgtgag cgggacacca gccggcggct gctggcggaa 1020 aaggagcggg agatggccga gatgcgggca aggatgcagc agcagctgga cgagtaccag 1080 gagcttctgg acatcaagct ggccctggac atggagatcc acgcctaccg caagctcttg 1140 gagggcgagg aggagaggct acgcctgtcc cccagcccta cctcgcagcg cagccgtggc 1200 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 <400> 3 cagtctagaa tggtgagcaa gggcgagga 29 <210> 4 <211> 31 <212> DNA <213> Artificial Sequence <400> 4 caggcggccg cttacatgat gctgcagttc t 31

Claims

1. A method for screening anti-aging drugs, characterized in that, include: Human primary skin fibroblasts overexpressing GFP-progerin protein were treated with the drug to be screened, and compared with cells overexpressing GFP-progerin protein that were not treated with the drug to be screened. The anti-aging effect of the drug to be screened was determined based on changes in cell morphology. The amino acid sequence of the GFP-progerin protein is shown in SEQ ID NO.1; The method of determining the anti-aging effect of the drug to be screened based on changes in cell morphology is as follows: When the localization of GFP-progerin protein in cells overexpressing GFP-progerin protein changes from being localized on the nuclear membrane to being localized in the cell nucleus after being treated with the drug to be screened, the drug to be screened is determined to have an anti-aging effect.

2. The screening method according to claim 1, characterized in that, The cells overexpressing GFP-progerin protein are primary cells overexpressing progerin protein.

3. The screening method according to claim 1, characterized in that, The cells overexpressing GFP-progerin protein were prepared in the following manner: The gene encoding the progerin protein was constructed into a lentiviral vector and then transfected into HK293T cells. The viral fluid was collected and then used to infect primary cells.

4. The screening method according to claim 3, characterized in that, The gene encoding the progerin protein includes a nucleotide sequence as shown in SEQ ID NO.

2.

5. The screening method according to claim 3, characterized in that, The lentiviral vector includes one or more of pCDH, pQCXIP, pLVX, or pLenti6 / TR.

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