Application of substance for detecting phase separation caused by SGF29 in cells in senescence detection

Abstract

The invention discloses application of a substance for detecting phase separation caused by SGF29 in cells in senescence detection. The invention finds that the SGF29 protein can be subjected to liquid-liquid phase separation along with the cell aging process, the phenomenon can be used as an index for detecting cell aging, the expression of the SGF29 protein can promote phase separation and cell aging, and the promotion can be prevented after mutation of the SGF29 protein. Evaluation indexes of cell senescence are expanded, meanwhile, cell senescence can be delayed by utilizing phase separation for destroying the SGF29 provided by the invention, and the phase separation can be used as a target spot for drug screening, so that disease treatment drugs with more accurate target spots and smaller side effects can be screened theoretically.

Description

Technical Field

[0001] The present invention relates to the application of a substance for detecting phase separation caused by SGF29 in cells in the field of biomedicine in detecting aging. Background Art

[0002] The global population is aging, and aging greatly increases the risk of disease in the elderly. It is estimated that by 2050, the total population aged 60 and above will reach 2.1 billion. The aging process is accompanied by the occurrence and development of a variety of diseases, such as cardiovascular and cerebrovascular diseases, diabetes, Alzheimer's disease, etc., which seriously threaten the life and health of the elderly. The increase in the elderly population is accompanied by many aging-related social and medical problems, which puts tremendous pressure on the country's economic development and medical health. Therefore, exploring the molecular mechanism of the aging process and finding ways to delay aging and improve aging-related diseases will make great contributions to social harmony, economic development and medical health.

[0003] Stem cell aging is a sign of the aging of the body and the development of age-related diseases. Currently, the pathogenesis of aging and aging-related diseases is not fully understood, and the prevention, diagnosis and treatment of aging and aging-related diseases have not yet achieved satisfactory results. It is urgent to develop new technologies and methods for the diagnosis, prevention and treatment of aging and aging-related diseases.

[0004] Cells are the basic units of structure and function of organisms. How the various components within cells gather at the correct time and space to perform their corresponding functions is a problem that cells need to solve in a series of basic life activities.

[0005] Cell senescence is a sign of the aging of the body and the development of age-related diseases. Currently, the commonly used methods for checking cell senescence include: telomere length determination, cell proliferation determination, oxidative stress determination, p21 and other protein level detection and analysis, DNA damage and repair assessment, SA-β-Gal staining and histological and morphological observation: HE staining, immunohistochemical staining and other methods can be used to observe the changes in tissue or cell morphology and composition to evaluate the aging state of cells. Hyman and Brangwynne proposed in 2009 that "phase separation" in cells can provide a specific way for specific molecules in cells to gather together, thereby forming a certain "order" inside the "chaotic" cells, providing a new idea for the problem that has troubled everyone for many years. Phase separation or phase change refers to the phenomenon that different components in cells collide and merge to form droplets, so that some components are encapsulated in the droplets and some components are blocked outside the droplets. Just like water and oil mixing, different components in cells will also separate from each other to form droplets. This is phase separation. In recent years, protein phase separation has been found to be widely present in cells, driving the formation of a variety of biomacromolecule aggregates that perform important biological functions. However, there are few reports on whether phase separation of biomacromolecules occurs during the aging process. Summary of the invention

[0006] The technical problem to be solved by the present invention is how to detect cell aging.

[0007] In order to solve the above technical problems, the present invention first provides the use of a substance for detecting phase separation in cells in preparing a product for detecting or assisting in detecting aging, or in detecting or assisting in detecting aging.

[0008] The phase separation (or phase change) refers to the process of forming an independent liquid phase in a solution (ie, liquid-liquid separation), and the formed liquid phase is a phase separation droplet (or phase change droplet).

[0009] The present invention also provides the use of a substance for detecting phase separation in cells in preparing a product for screening or assisting in screening senescent cells, or in screening or assisting in screening senescent cells.

[0010] In the above application, the phase separation may be phase separation caused by SGF29.

[0011] The substance for detecting the phase separation caused by SGF29 may be a substance that specifically recognizes SGF29, such as an SGF29 antibody.

[0012] The present invention also provides a substance for detecting the amount of SGF29 gene expression in cells, or the use of a substance for detecting the content of SGF29 in cells in preparing a product for detecting or assisting in detecting aging, or in detecting or assisting in detecting aging.

[0013] In the above application, the substance used to detect the SGF29 content in the cells can be a substance that specifically recognizes SGF29, such as an SGF29 antibody.

[0014] In the above, the aging may be the aging of animal cells, tissues, organs or individuals. The cells may be any somatic cells of an animal, including stem cells (such as mesenchymal stem cells), non-stem cells (such as cancer cells), and the like.

[0015] The present invention also provides a method for preparing a senescent cell model, the method comprising: increasing the content or activity of SGF29 in a receptor cell, or promoting the expression of an SGF29 gene in a receptor cell, or promoting the phase separation of SGF29 in a receptor cell, to obtain a senescent cell.

[0016] In the above method, the SGF29 phase separation in the recipient cell refers to the phase separation caused by SGF29 in the cell.

[0017] In the above method, the cell can be any animal cell, including stem cells, cancer cells, etc. In one embodiment of the present invention, the cell is a mesenchymal stem cell or a 293T cell.

[0018] The above method can be achieved by introducing the SGF29 gene into the recipient cell and expressing the SGF29 gene.

[0019] The present invention also provides the use of a substance that inhibits SGF29 gene expression or knocks out SGF29 gene in the preparation of a product for treating and / or preventing and / or delaying aging, or in treating and / or preventing and / or delaying aging.

[0020] In the above application, the substance that inhibits SGF29 gene expression or knocks out SGF29 gene can be a substance that specifically inhibits SGF29 gene expression, such as siRNA, etc., or it can be a substance that edits SGF29 gene through gene editing (such as crisper / cas9 system, etc.).

[0021] The substance may be a substance that replaces the 207th arginine codon encoding the SGF29 protein in the SGF29 gene with 1 proline codon.

[0022] The present invention also provides the use of a substance that reduces the content or activity of SGF29 in the preparation of a product for treating and / or preventing and / or delaying aging, or in treating and / or preventing and / or delaying aging.

[0023] The present invention also provides the use of a substance that inhibits phase separation caused by SGF29 in cells in the preparation of a product for treating and / or preventing and / or delaying aging, or in treating and / or preventing and / or delaying aging.

[0024] The substance that inhibits the phase separation caused by SGF29 in cells can be any substance that can inhibit phase separation in cells (such as 1,6-hexanediol, NaCl, PEG-4000, etc.), and can also be a substance that specifically inhibits the phase separation caused by SGF29. The substance that specifically inhibits the phase separation caused by SGF29 can be a substance that specifically recognizes SGF29, such as SGF29 antibodies, etc.

[0025] In the above applications, the aging may be the aging of animal cells, tissues, organs or individuals. The cells may be any somatic cells of an animal, including stem cells (such as mesenchymal stem cells), non-stem cells (such as cancer cells), etc.

[0026] The present invention finds that SGF29 protein can undergo liquid-liquid phase separation during the cell aging process, and this phenomenon can be used as an indicator for detecting cell aging. The expression of SGF29 protein can promote phase separation and cell aging, and mutation of SGF29 protein can prevent this promotion. Compared with the prior art, the present invention greatly expands the evaluation indicators of cell aging. At the same time, the destruction of SGF29 phase separation proposed in the present invention can delay cell aging and use it as a target for drug screening. In theory, it is possible to screen disease treatment drugs with more precise targets and fewer side effects.

[0027] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The experimental result diagram of Example 1. Among them, the left side of Figure A is the SA-β-gal staining test diagram of the aging marker of human mesenchymal stem cells in the young group and the old group, and the right side is the statistical result; B is the test and statistical results of the monoclonal formation ability of human mesenchymal stem cells in the young group and the old group; C is the test results of the nuclear-cytoplasmic separation experiment of human mesenchymal stem cells in the young group and the old group; D is the immunofluorescence detection of SGF29 in the young group and the old group human mesenchymal stem cells to form phase-change droplets, the left figure is the photo result, and the right figure is the statistical fluorescence intensity of the SGF29 of the line (arrow and its tail) of the cells in the young group and the old group. SGF29 in the old group cells forms a dot structure, so the SGF29 at the line has a high-low fluorescence intensity curve, on the contrary, the young group cells do not form an obvious dot structure, and the fluorescence intensity curve of SGF29 at the line has a small fluctuation range, and the abscissa of the right figure represents the fluorescence intensity of the SGF29 protein in the cell nucleus; E is the statistical number of phase-change droplets formed by SGF29 in each cell of the human mesenchymal stem cells in the young group and the old group.

[0029] Figure 2 The experimental result diagram of Example 2. Among them, A is the result of detecting EGFP-SGF29 to form phase transition droplets in HEK293T cells transfected with EGFP lentivirus and EGFP-SGF29 lentivirus respectively; B is the result of further 3D reconstruction of EGFP-SGF29 forming phase transition droplets in HEK293T cells; C is the result of detecting EGFP-SGF29 to form phase transition droplets in human stem cell lentivirus infection with EGFP lentivirus and EGFP-SGF29 lentivirus. The left picture is the photo result, and the right picture is the statistical fluorescence intensity of the green fluorescence of the line (arrow and its tail) of the cells transfected with EGFP lentivirus and EGFP-SGF29 lentivirus. The green fluorescence in the EGFP-SGF29 group cells formed a dot-like structure. Therefore, the green at the line showed a high-low fluorescence intensity curve, while the EGFP group cells did not form There is an obvious dot-like structure, and the green fluorescence intensity curve at the dashed point has a small fluctuation range. The horizontal axis of the right figure represents the green fluorescence intensity; D is that 10% 1,6-hexanediol can destroy the phase-separated phase-change droplets of EGFP-SGF29; E is the change of EGFP-SGF29 phase-change droplets after mesenchymal stem cells are infected with EGFP-SGF29 lentivirus, whether fusion occurs; F is the fluorescence photobleaching recovery imaging of EGFP-SGF29 phase-change droplets after fluorescence photobleaching after mesenchymal stem cells are infected with EGFP-SGF29 lentivirus to observe whether the EGFP-SGF29 phase-change droplets recover after fluorescence photobleaching. The results show that after fluorescence photobleaching of EGFP-SGF29, SGF29 can be observed to recover (right figure, showing the change in fluorescence intensity of the green fluorescent droplets in the dotted circle in the left figure), further indicating that the phase-change droplets formed by EGFP-SGF29 have the characteristics of phase separation.

[0030] Figure 3 The experimental results of step 2 of Example 3 are shown in Figure A. A shows the phase separation of 5 μM in vitro purified 6×His-SGF29 protein; B shows the precipitation experiment of SGF29 phase separation analyzed by SDS-PAGE and Coomassie blue staining, where P represents precipitation and S represents supernatant.

[0031] Figure 4 The experimental results of Example 4 are shown in Figure A. A is the predicted score of SGF29 disorder and irregularity using the PONDR program and the crystal structure of SGF29; B is the protein level of EGFP-SGF29 or EGFP-SGF29 R207P expressed in human mesenchymal stem cells by Western blot analysis; C and D are the changes in the ability of phase transition after expressing EGFP-SGF29WT or R207P mutant in mesenchymal stem cells; E is a fluorescence photobleaching recovery experiment performed on mesenchymal stem cells infected with lentivirus expressing EGFP-SGF2 or EGFP-SGF29-R207P.

[0032] Figure 5 The experimental results of Example 5. A is the SA-β-gal staining result after young human mesenchymal stem cells were infected with EGFP, EGFP-SGF29 or EGFP-SGF29 R207P lentivirus; B is the monoclonal formation ability detection result of young human mesenchymal stem cells infected with EGFP, EGFP-SGF29 or EGFP-SGF29 R207P lentivirus; C is the Ki6 positive cell detection result of young human mesenchymal stem cells infected with EGFP, EGFP-SGF29 or EGFP-SGF29 R207P lentivirus.

[0033] Figure 6 The experimental results of Example 6. A is the ChIP-qPCR experimental results of young mesenchymal stem cells expressing Flag-EGFP-SGF29 or Flag-EGFP-SGF29R207P; B is the result of DNA FISH, red is the DNA FISH signal of CDKN1A gene promoter, because the cell allele has two signal points of CDKN1A promoter, green is the fluorescence signal of SGF29, SGF29 forms a dot structure; C is the detection result of CDKN1A mRNA expression level after expressing EGFP-SGF29 or EGFP-SGF29 R207P in young human mesenchymal stem cells; D is the p21 of young mesenchymal stem cells expressing Flag-EGFP-SGF29 or Flag-EGFP-SGF29 R207P. Cip1 A is the result of protein expression level detection; E is the result of western blot after CDKN1A expression was inhibited by siRNA in mesenchymal stem cells expressing EGFP or EGFP-SGF29; F is the result of SA-β-gal staining after CDKN1A expression was inhibited by siRNA in mesenchymal stem cells expressing EGFP or EGFP-SGF29.

[0034] Figure 1-Figure 6 In the table, * indicates significant analysis p < 0.05, ** indicates significant analysis p < 0.01, and *** indicates significant analysis p < 0.001. DETAILED DESCRIPTION

[0035] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions. The materials, reagents, instruments, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources. The quantitative tests in the following examples were all repeated at least three times, and the results were averaged. In the following examples, unless otherwise specified, the first position of each nucleotide sequence in the sequence table is the 5' terminal nucleotide of the corresponding DNA / RNA, and the last position is the 3' terminal nucleotide of the corresponding DNA / RNA.

[0036] Example 1: Mesenchymal stem cell aging detection SGF29 forms phase transition droplets

[0037] 1. Directed differentiation of human embryonic stem cells into human mesenchymal stem cells

[0038] (1) hESCs (human embryonic stem cell H9 cell line (WiCell product, catalog number: WA09(H9)-DL-7)) were passaged on feeder cells MEF, and fresh culture medium was replaced every day to keep the cells in good condition. Contact between hESCs clones was avoided.

[0039] (2) After about 3-4 days of passaging, rinse the cells once with DMEM / F12 medium, add an appropriate amount of 0.5 mg / mL dispase digestion solution, and place them in a 37°C, 5% CO2 cell culture incubator for about 20 minutes to allow the hESCs clones to transition from an adherent state to a suspended state.

[0040] (3) Wash 2-3 times with DMEM / F12 medium to remove the dispase digestion solution. Then resuspend in CDF12 medium containing 4 ng / mL FGF2 and inoculate in a low-adhesion 6-well plate. Change the medium on the second day and every other day thereafter.

[0041] The components of CDF12 medium are as follows:

[0042] DMEM / F12 medium (Gibco, 11330057) 500mL;

[0043] Knockout Serum Replacement (Gibco, 10828028) 130mL;

[0044] MEM NEAA (100×, Gibco, Cat.#11140-050) 5.8mL;

[0045] Penicillin streptomycin (100×, Gibco, Cat.#15140-122) 5.8mL;

[0046] Plasmocin TM Treatment (InvivoGen, Cat.#ant-mpt) 50μL;

[0047] bFGF (Joint Protein Central, 13256029) 130 μL;

[0048] 55 μM β-Mercaptoethanol (Gibco, 21985023) 630 μL.

[0049] (4) After 3 days, rinse the hESCs clones once with MEM a (GIBCO, 32571-101), then inoculate them into a 6-well plate pre-coated with Matrigel, add hMSC differentiation medium (hMSC-DM), and change the medium every other day. The hESCs clones will adhere to the 6-well plate, and spindle-shaped cells will gradually grow out of the hESCs clones.

[0050] Among them, hMSC differentiation medium (hMSC-DM) is as follows:

[0051] MEM a(GIBCO,32571-101)500mL;

[0052] Fetal bovine serum (Gibco, Cat.#10270-106, Lot.#42Q8982K) 56mL;

[0053] GlutaMAX TM -I (100×, Gibco, Cat.#35050-061)5.8mL;

[0054] MEM NEAA (100×, Gibco, Cat.#11140-050) 5.8mL;

[0055] Penicillin streptomycin (100×, Gibco, Cat.#15140-122) 5.8mL;

[0056] Plasmocin TM Treatment (InvivoGen, Cat.#ant-mpt) 50μL;

[0057] bFGF (Joint Protein Central, 13256029) 130 μL;

[0058] 5ng / mL TGF-β50μL.

[0059] (5) After 10 days of differentiation, the cells were digested with TrypLE and 2 × 10 5 The cells were seeded in a 6-well plate pre-coated with Matrigel, and hMSC culture medium was added. The medium was changed every 3 days, and small fibrous cells gradually appeared.

[0060] The components of hMSC culture medium are as follows:

[0061] MEM a(GIBCO,32571-101)500mL;

[0062] Fetal bovine serum (Gibco, Cat.#10270-106, Lot.#42Q8982K) 56mL;

[0063] GlutaMAX TM -I (100×, Gibco, Cat.#35050-061)5.8mL;

[0064] MEM NEAA (100×, Gibco, Cat.#11140-050) 5.8mL;

[0065] Penicillin streptomycin (100×, Gibco, Cat.#15140-122) 5.8mL;

[0066] bFGF (Joint Protein Central, 13256029) 13μl;

[0067] (6) Plasmocin TM Treatment (InvivoGen, Cat.#ant-mpt) 50μL.

[0068] (6) After 5-10 days of culture, cells that are about to grow dense are digested with TrypLE and 1×10 6 The cells were seeded in two 10 cm culture dishes pre-coated with 0.5% gelatin, and hMSC culture medium was added, with the medium changed every 2 days.

[0069] (7) After 3-5 days, the cells will become dense and ready for flow cytometry sorting. Prepare FACS buffer (10% FBS, diluted in PBS), PE anti-CD73 antibody (BD, Cat.#550257, 1:100 dilution), FITC anti-CD90 antibody (BD, Cat.#555595, 1:200 dilution), APC anti-CD105 antibody (BioLegend, Cat.#323208, 1:200 dilution).

[0070] (8) Rinse the cells with PBS, digest the cells with TrypLE, collect the cells, stain with trypan blue, and count them with a hemocytometer. Take out 1×10 7 Centrifuge the cells at room temperature, 1000 rpm for 5 minutes. Discard the supernatant, resuspend the cells with 1 mL FACS buffer, and add CD73 / 90 / 105 antibodies. Wrap with tin foil and incubate for 30 minutes at room temperature in the dark. To improve the efficiency of antibody staining, shake the cells every 5-10 minutes. Centrifuge at room temperature, 1000 rpm for 5 minutes, discard the supernatant, and resuspend the cells with 0.5-1 mL FACS buffer.

[0071] (9) CD73 / CD90 / CD105 triple-positive cells were sorted and collected using BD flow cytometer FACSAria IIIu.

[0072] (10) After flow sorting, count the cells using a blood cell counting plate, using 1×10 6 Plant the cells in a 10 cm culture dish pre-coated with 0.1% gelatin, add hMSC culture medium, and change the medium every 2 days. To prevent contamination by the sorted cells, the concentration of penicillin streptomycin in the culture medium can be doubled, and plasmosin can also be added.

[0073] (11) When the cells were about to grow dense, passage 2 (P2) and P3 (the cells sorted back were P1) hMSCs were cultured at 5×10 5 / tube and stored in liquid nitrogen until use.

[0074] Mesenchymal stem cells from young and old people were selected for further experiments. The human mesenchymal stem cells from the young group were hMPCs of generation P5 (P5_hMPCs); the human mesenchymal stem cells from the old group were hMPCs of generation P15 (P15_hMPCs).

[0075] 2. Mesenchymal stem cell culture

[0076] Human mesenchymal stem cells (hMSCs) derived from human embryonic stem cells (hESCs) were cultured in mesenchymal stem cell medium. Mesenchymal stem cell medium (MSC medium): MEM a (GIBCO, 32571-101) supplemented with 10% fetal bovine serum (FBS) (Cat#10099-141, Lot#1616964, Thermo Fisher Scientific), 0.1 mM NEAAs (Thermo Fisher Scientific), 1% penicillin / streptomycin (Thermo Fisher Scientific) and 1 ng / mL bFGF (Joint Protein Central, Incheon, South Korea).

[0077] 3. SA-β-Gal staining experiment

[0078] 1) When the density of cells on the cell culture plate reaches 70%, staining experiments can be started.

[0079] 2) After removing the culture medium, wash the cells twice with PBS, then add fixation buffer (2% formaldehyde and 0.2% glutaraldehyde) and let stand for 5 minutes at room temperature.

[0080] 3) After fixation, wash twice with PBS, then add fresh staining solution and stain overnight at 37°C. Wash twice with PBS the next day, randomly select fields in each well, and use ImageJ software to count the percentage of SA-β-gal positive cells. The staining process should be carried out in the dark. Three biological replicates were set for each cell line, and each experiment was repeated at least twice independently. Statistical significance was evaluated by two-tailed unpaired Student's t-test.

[0081] The staining solution formula is as follows: NaCl, 150mM; MgCl2, 2mM; X-gal, 1mg / mL; K3[Fe(CN)6], 100mM; K4[Fe(CN)6]·3H2O, 100mM; citric acid / sodium phosphate buffer (pH=6.0), 1mg / mL; ddH2O, supplemented to 20mL.

[0082] The results are as follows Figure 1 As shown in A, the results showed that the human mesenchymal stem cells in the elderly group had darker SA-β-gal staining and a higher proportion of SA-β-gal positive cells, indicating that the elderly mesenchymal stem cells used were senescent cells.

[0083] 4. Monoclonal formation experiment

[0084] 1) The cells were seeded into 6-well cell culture plates at a cell density of 6,000 cells / well, and MSC culture medium was added and replaced every 2 days. The cells were cultured for 9-12 days until the cells grew to a density suitable for the experiment.

[0085] 2) Wash twice with PBS, fix with 4% paraformaldehyde fixative and stain with 0.2% crystal violet staining solution and leave at room temperature for 1 hour for staining.

[0086] 3) The stained cell culture plates were scanned by a scanner, and the cell density was further measured by ImageJ software (NIH). Three biological replicates were set for each group of cells, and each experiment was repeated at least twice independently. Statistical significance was evaluated by a two-tailed unpaired Student's t-test.

[0087] The results are as follows Figure 1 As shown in B, the results showed that the human mesenchymal stem cells in the aged group were lighter in crystal violet staining and had relatively weaker clone-forming ability, further indicating that the aged mesenchymal stem cells used were senescent cells.

[0088] 5. Nuclear-Cytoplasmic Separation Experiment

[0089] 1) Wash 1×10 7 Live hMSC cells were isolated and lysed in cytoplasmic lysis buffer (10 mM KCl, 10 mM HEPES-KOH pH 7.9, 1.5 mM MgCl2, 1 mM DTT, 0.4% NP-40 and 1× protease inhibitor cocktail (Roche)) and rotated at 4°C for 10 min.

[0090] 2) After step 1), centrifuge at 15000×g for 5 minutes at 4°C, and save the soluble supernatant (referred to as cytoplasmic protein solution) for detecting cytoplasmic proteins. The precipitated nuclei were washed with ice-cold PBS, then resuspended in nuclear lysis buffer (420mM NaCl, 20mM HEPES-KOH pH 7.9, 0.2mM EDTA, 0.1mM EGTA, 25% glycerol, 1mM DTT and 1× protease inhibitor cocktail) and vortexed at 4°C for 1 hour. After centrifugation at 15000×g for 15 minutes, the supernatant was collected and labeled as nuclear protein solution.

[0091] Western blot was used to detect the protein expression level of SGF29 in cytoplasmic protein solution and nuclear protein solution, and the relative expression level was calculated. The cytoplasmic marker was β-tubulin and the nuclear marker was TAP1. The primary antibodies used were SGF29 antibody (SantaCruz, sc-515286), tubulin antibody (Immunoway, YM3030), TAP1 antibody (SantaCruz, sc-32319), and the secondary antibodies used were HRP-conjugated goat anti-Mouse lgG (H+L) (ZSGB-Bio, ZB2305) and HRP-conjugated goat anti-Rabbit lgG (H+L) (ZSGB-Bio, ZB2301).

[0092] The results are as follows Figure 1 As shown in C, the results show that only the corresponding markers can be detected in the cytoplasm and the nucleus. In addition, it can be seen that the SGF29 in the cytoplasm of human mesenchymal stem cells in the elderly group decreased, while the SGF29 in the nucleus increased significantly, indicating that the nuclear protein level of SGF29 in human mesenchymal stem cells in the elderly group increased.

[0093] VI. Cell Immunofluorescence Staining Experiment

[0094] 1) Gelatin-coated culture plates were equilibrated in a 37°C carbon dioxide cell culture incubator overnight.

[0095] 2) 5×10 4 or 2.5 × 10 4 hMSC cells were cultured continuously until the cell confluence reached about 80% for the next staining experiment.

[0096] 3) After step 2) was completed, the culture medium was removed and the cells were washed twice with PBS. 500 μL of 4% paraformaldehyde fixative was added to each well to fix the cells for 30 min at room temperature, and then washed twice with PBS.

[0097] 4) After discarding PBS, add 500 μL of cell permeabilization solution (0.4% Triton X-100 diluted in PBS) to the culture wells, let stand at room temperature for about 30 minutes, and then wash twice with PBS.

[0098] 5) Discard PBS, add blocking solution (donkey serum (abcam, ab7475) diluted in PBS, donkey serum volume percentage is 10%) to the culture wells, and let stand at room temperature for 1 hour. Cover slips were placed in 24-well cell culture plates one day in advance, sterilized by UV for more than 30 minutes, and treated with 0.1% gelatin for 16 hours.

[0099] 6) Aspirate the blocking solution, carefully transfer the coverslips to a humidified chamber, drop the diluted primary antibody (SGF29 antibody (SantaCruz, sc-515286); Cellular Senescence Detection Kit-SPiDER-βGal dye (DOJINDO Laboratories, Cat#PL507) on the surface of the coverslips, and incubate the primary antibody and dye overnight at 4°C.

[0100] 7) The next day, the primary antibody was removed and the samples were washed three times with PBS. Then, diluted immunofluorescence secondary antibody (Goat anti-Mouse IgG1 Cross-Adsorbed Secondary Antibody, Alexa Fluor488 (Invitrogen, A21121)) and DNA dye (Hoechst33342) were added to the surface of coverslips and incubated with secondary antibody at room temperature for 1 hour.

[0101] 8) Remove the secondary antibody, wash the cells three times with PBS, and then carefully invert the coverslips onto the slides. Anti-fluorescence quenching agent has been added to the slides in advance.

[0102] 9) Observe the staining effect under a laser confocal microscope and take photos for statistics.

[0103] The results are as follows Figure 1 As shown in D and E, the results show that the SGF29 phase transition droplets (i.e., point-like structures) formed by the human mesenchymal stem cells in the aged group are larger and have a stronger fluorescence intensity, indicating that SGF29 forms aging-related phase transition droplets in the human mesenchymal stem cells in the aged group; the number of phase transition droplets shows that the human mesenchymal stem cells in the aged group form more structures, further indicating that SGF29 has a stronger ability to form aging-related phase transition droplets in the human mesenchymal stem cells in the aged group, indicating that the phase separation of SGF29 is related to the aging of human mesenchymal stem cells.

[0104] Example 2: Detection of EGFP-SGF29 in 293T cells and mesenchymal stem cells to form phase transition droplets

[0105] The culture medium used is as follows: HEK293T culture medium composition is as follows:

[0106] HyClone DMEM / High glucose (Cytiva, Cat.#SH30243.01) 500mL;

[0107] Fetal bovine serum (Gibco, Cat.#10270-106, Lot.#42Q8982K) 56mL;

[0108] GlutaMAX TM -I (100×, Gibco, Cat.#35050-061)5.8mL;

[0109] MEM NEAA (100×, Gibco, Cat.#11140-050) 5.8mL;

[0110] Penicillin streptomycin (100×, Gibco, Cat.#15140-122) 5.8mL;

[0111] Plasmocin TM Treatment (InvivoGen, Cat.#ant-mpt) 50μL.

[0112] hMSC culture medium components are as follows:

[0113] MEM a(GIBCO,32571-101)500mL;

[0114] Fetal bovine serum (Gibco, Cat.#10270-106, Lot.#42Q8982K) 56mL;

[0115] GlutaMAX TM -I (100×, Gibco, Cat.#35050-061)5.8mL;

[0116] MEM NEAA (100×, Gibco, Cat.#11140-050) 5.8mL;

[0117] Penicillin streptomycin (100×, Gibco, Cat.#15140-122) 5.8mL;

[0118] bFGF (Joint Protein Central, 13256029) 13μl;

[0119] Plasmocin TM Treatment (InvivoGen, Cat.#ant-mpt) 50μL.

[0120] 1. Lentivirus packaging

[0121] In this example, Lipofectamine TM 3000 Transfection Kit (Invitrogen, Cat.#L3000-015) was used to package lentivirus, and the steps were as follows:

[0122] 1) Inoculate HEK293T cells in a 10 cm culture dish, add 10 mL of HEK293T culture medium and place in a 37°C, 5% CO2 cell culture incubator for static culture.

[0123] 2) When the density of HEK293T cells reaches 95-99%, you can start packaging lentivirus.

[0124] Prepare component A according to the following components and dosage:

[0125] I reduced serum media (Gibco, Cat. #31985-070) 1.5mL;

[0126] pLenti expression vector 7.5μg;

[0127] psPAX2 5 μg;

[0128] pMD2G 2.5 μg;

[0129] P3000 TM Reagent (Invitrogen, Cat. #100022058) 25 μL.

[0130] Prepare component B according to the following components and dosage:

[0131] I Reduced Serum Media (Gibco, Cat. #31985-070) 1.5mL;

[0132] Lipofectamine TM 3000Reagent (Invitrogen, Cat. #100022052) 25 μL.

[0133] Add component A dropwise into component B, mix well, and let stand at room temperature for 10-20 minutes to obtain a mixed solution of components A / B.

[0134] 3) Remove 4 mL of culture medium from the 10 cm culture dish, then slowly drop the A / B component mixture into the remaining culture medium and shake gently to mix.

[0135] 4) Place in a 37°C, 5% CO2 cell culture incubator for 6 hours.

[0136] 5) The culture medium was discarded, fresh HEK293T culture medium was added, and the cells were placed in a 37° C., 5% CO 2 cell culture incubator for further culture.

[0137] 6) After 24 h, collect all the culture medium (i.e., all liquid except cells) into a 50 mL centrifuge tube and temporarily store in a 4°C refrigerator. Then, add fresh HEK293T culture medium to the cells and continue culturing in a 37°C, 5% CO2 cell culture incubator.

[0138] 7) After 52 hours, collect the culture medium for the second time into a 50 mL centrifuge tube and mix the two collected culture media together, about 40 mL.

[0139] 8) The mixture of the two culture media was centrifuged at room temperature and 2000 rpm for 10 min to remove floating cells and cell debris, and the supernatant was collected.

[0140] 9) Filter the supernatant once using a 0.45 μm filter membrane.

[0141] 10) The obtained filtrate was centrifuged at 4°C, 19400 rpm for 2 h to concentrate the virus.

[0142] 11) The supernatant was discarded, and an appropriate volume of HEK293T culture medium was added to resuspend the virus (i.e., EGFP-SGF29 lentivirus) to obtain an EGFP-SGF29 lentivirus suspension, which was aliquoted and stored in a -80°C refrigerator.

[0143] Among them, pLenti expression vector is a recombinant plasmid obtained by replacing the DNA fragment between the BamHI and MluI recognition sequences of the pLE4 vector with the SGF29 gene (whose sequence is shown in sequence 1 in the sequence list). pLentiexpression vector can express a fusion protein formed by SGF29 (whose sequence is shown in sequence 2 in the sequence list) and EGFP (denoted as EGFP-SGF29).

[0144] pLE4 vector: gifted by Dr. Tomoaki Hishida, described in the literature “Huifang Hu et al., ZKSCAN3 counteracts cellular senescence by stabilizing heterochromatin., Nucleic Acids Research, 20, doi:10.1093 / nar / gkaa425”.

[0145] psPAX2: Addgene product, #12260.

[0146] pMD2G: Addgene product, #12259.

[0147] According to the above method, pLenti expression vector was replaced with pLenti expression vectorR207P, and other steps remained unchanged, and the obtained virus was recorded as EGFP-SGF29 R207P lentivirus. pLenti expression vector R207P is a recombinant plasmid obtained by replacing the DNA fragment between the BamHI and MluI recognition sequences of the pLE4 vector with the SGF29 R207P gene (whose sequence is shown in sequence 3 in the sequence list). pLenti expression vector can express a fusion protein formed by SGF29R207P (whose sequence is shown in sequence 4 in the sequence list) and EGFP (referred to as EGFP-SGF29R207P).

[0148] According to the above method, the pLenti expression vector was replaced with the pLE4 vector, and the other steps remained unchanged. The obtained virus was recorded as EGFP lentivirus.

[0149] 2. Lentivirus titer determination

[0150] 1) Before lentiviral transfection of hMSCs, the titer of lentivirus should be determined. Human mesenchymal stem cells (P5_hMPCs) were cultured at 5×10 4 Each well was inoculated in a 24-well plate pre-coated with 0.1% gelatin.

[0151] 2) When the cells grow to an appropriate density, add 10 mg / mL polybrene solution to the hMSC culture medium at a dilution ratio of 1:1000 and mix well.

[0152] 3) Set a lentiviral transfection titer gradient, such as 0 / 0.1 / 0.2 / 0.4 / 0.8 / 1.6 μL / well (24-well plate), mix well, and place in a 37° C., 5% CO2 cell culture incubator.

[0153] 4) After culturing for 24 hours, the lentivirus successfully infected the cells, the culture medium was discarded, fresh cell culture medium was added, and the cells were placed in a 37° C., 5% CO 2 cell culture incubator for further culturing for 24 hours.

[0154] 5) At this time, the cells in the 24-well plate are almost dense and the protein has been expressed. Through fluorescence detection in the 488nm fluorescence channel, the virus titer with a fluorescence positive rate of fusion tag protein close to 100% is selected as the appropriate titer for subsequent transfection of cells.

[0155] 3. Lentiviral transfection of human mesenchymal stem cells and 293T cells

[0156] Cells used: young human mesenchymal stem cells (P5_hMPCs) and HEK293T cells; viruses used are the EGFP-SGF29 lentivirus and EGFP lentivirus in step one.

[0157] 1) Cells were cultured at 1×10 5 Each well was inoculated in a 6-well plate pre-coated with 0.1% gelatin.

[0158] 2) When HEK293T cells or human mesenchymal stem cells grow to an appropriate density, add 10 mg / mL polybrene solution to the culture medium (HEK293T cells use HEK293T culture medium, human mesenchymal stem cells use human mesenchymal stem cell culture medium) at a dilution ratio of 1:1000 and mix well.

[0159] 3) According to the appropriate virus titer determined previously, add the corresponding volume of lentivirus, mix well, and place in a 37°C, 5% CO2 cell culture incubator.

[0160] 4) After culturing for 24 hours, the lentivirus successfully infects the cells, the culture medium is discarded, fresh cell culture medium is added, and the cells are placed in a 37° C., 5% CO 2 cell culture incubator for further culturing to obtain transfected cells.

[0161] 5) Before the cells grow dense, perform cell subculture on a clean bench designed for virus-related experiments in preparation for related experiments.

[0162] The results are as follows Figure 2 As shown in Figures AC, HEK293T cells were transfected with EGFP lentivirus and EGFP-SGF29 lentivirus, respectively. The results showed that a large number of phase transition droplets could be observed in cells expressing EGFP-SGF29, indicating that EGFP-SGF29 has the ability to form phase separation ( Figure 2 A); further using confocal imaging software to perform 3D reconstruction of the phase transition droplets formed by EGFP-SGF29 in HEK293T cells, the spherical structure of EGFP-SGF29 can be observed, further indicating that EGFP-SGF29 forms phase separation phase transition droplets in HEK293T cells ( Figure 2 In human MSCs, EGFP and EGFP-SGF29 lentiviruses were infected respectively. The results showed that EGFP-SGF29 could also be observed to form phase transition droplets in human MSCs ( Figure 2 Middle C).

[0163] 4. Chemical treatment

[0164] Phase separation is a common phenomenon in cells. Thousands of proteins or nucleic acid molecules form membrane-less "compartments" (i.e., phase separation droplets) inside complex cells, just like oil droplets in water. They participate in many important biological processes in cells, such as transcription regulation, stress, protein quality control, DNA replication, etc. without interfering with each other. 1,6-Hexanediol (1,6-HD) is a small chemical molecule that can dissolve phase separation droplets.

[0165] 1) Dissolve 1,6-hexanediol (Sigma-Aldrich, 240117) in PBS to obtain a 10% 1,6-hexanediol solution.

[0166] 2) Human mesenchymal stem cells (P5_hMPCs) were cultured at 1×10 5 Each well was inoculated with 4 glass coverslips and cultured for 24 hours.

[0167] 3) Then, the mesenchymal stem cells were infected with EGFP-SGF29 lentivirus. After 96 hours, the infected cells were placed in a 10% 1,6-hexanediol solution for 1 minute. The cells not treated with 1,6-hexanediol were used as controls.

[0168] 4) Then, the cells were immediately fixed with 4% PFA fixative and fluorescence detection was performed using the 488 nm fluorescence channel.

[0169] Results Figure 2 In middle D, the results show that 1,6-hexanediol can destroy the phase separation phase transition droplets of EGFP-SGF29, further indicating that SGF29 forms phase separation in human mesenchymal stem cells.

[0170] 5. Live Cell Imaging

[0171] For live cell imaging, human mesenchymal stem cells (P5_hMPCs) infected with EGFP-SGF29 lentivirus for 48 hours were plated at 1 × 10 cells / well. 5 The cells were seeded at a density of 1000 cells / mL on a confocal live cell imaging dish (Cellvis, D35-20-1.5-N). After 20 h, live cell imaging was performed using an Andor Dragonfly 505 fluorescence microscope with a temperature-controlled heating system, with images collected every 2.5 min.

[0172] Results Figure 2 Middle E, the results show that EGFP-SGF29 phase transition droplets can fuse.

[0173] 6. Fluorescence recovery after photobleaching (FRAP)

[0174] For FRAP experiments, young human mesenchymal stem cells (P5_hMPCs) infected with EGFP-SGF29 lentivirus or EGFP-SGF29 R207P lentivirus for 96 hours were plated on confocal live cell imaging culture dishes (FluoroDish, WorldPrecision Instruments) and imaged using a Zeiss LSM900 confocal fluorescence microscope. Fluorescence bleaching was performed using a 488nm laser at 95% power. A circular region of interest (ROI) with a radius of 1.5μm was defined around a single SGF29 phase transition droplet. Image acquisition was performed every second, and the fluorescence intensity of the spot was finally measured using ImageJ software.

[0175] The results are as follows Figure 2 As shown in F, the results show that after fluorescence bleaching, SGF29 recovery can be observed, further indicating that the phase transition droplets formed by EGFP-SGF29 have the characteristics of phase separation.

[0176] Example 3: Phase separation of recombinant protein SGF29 in vitro

[0177] 1. Recombinant protein purification

[0178] 1) The DNA fragment between the XbaI and BamHI recognition sequences of the pET28a vector was replaced with the SGF29 gene (whose sequence is shown in sequence 1 in the sequence table), and the resulting recombinant plasmid was recorded as pET28a-SGF29. pET28a-SGF29 can express a fusion protein formed by SGF29 (whose sequence is shown in sequence 2 in the sequence table) and a 6×His tag (recorded as 6×His-SGF29).

[0179] pET28a-SGF29 was introduced into Escherichia coli Rosset 2 (DE3) (Solarbio, C1420) to obtain the recombinant bacteria Rosset / pET28a-SGF29.

[0180] The recombinant Rosset / pET28a-SGF2 was cultured at 37°C for 8 hours and the OD 600 Isopropyl-β-d-1-thiogalactopyranoside (IPTG) was added at a final concentration of 1 mM between 0.5 and 10, and induced overnight at 28°C.

[0181] 2) Collecting the bacteria. After step 1), the obtained bacterial solution was centrifuged at 4000 rpm and 4°C for 20 min, the supernatant medium was discarded, and the bacteria were washed once with PBS.

[0182] 3) Dissolve the cell wall. After step 2), resuspend the cells in 100 ml PBS, add PMSF and lysozyme at a ratio of 1:100, and shake at room temperature for 10 minutes.

[0183] 4) Lyse cells. After step 3), add urea lysis solution to 300 ml, mix well and sonicate twice (4s; 6s; 4min), alternately to prevent overheating. When the bacterial solution is clear, take 160 μl of sample (record this sample as cell lysis solution), and centrifuge the remaining sample at 8000 rpm for 10 min.

[0184] 5) Treat Ni column. Resuspend the stored beads and mix well. Take 6 ml and centrifuge at 500 rpm for 10 min. Discard the supernatant and wash three times with urea lysis buffer to remove ethanol. After washing, soak in urea lysis buffer and place at 4°C for later use.

[0185] 6) Beads binding. Transfer the supernatant after centrifugation in step 4) to a new centrifuge tube, add Beads and bind at 4°C for at least 30 minutes. Resuspend the precipitate after centrifugation in step 4) with 1 ml ddH2O, and take 160 μl of the sample (record this sample as the cell lysis precipitate).

[0186] 7) Elute the impurities. After step 6) is completed, pour the liquid that has combined with the beads into the filter, connect a centrifuge tube below (to prevent insufficient binding of the beads and waste of protein) to collect the flow through (i.e., the outflow liquid), and take 160μl as a sample. Wash the beads 5 times with wash buffer, resuspend with a pipette to fully elute the impurities, collect the eluate in a centrifuge tube, and take 160μl as a sample.

[0187] 8) Elute the target protein. Prepare several EP tubes, mark them with numbers, and elute the target protein with Elute buffer. Collect 1 ml in each EP tube, collect at least 9 tubes, determine the protein concentration, and take the 4 tubes with the highest concentration, 20 μl in each tube. Purified 6×His-SGF29 is obtained.

[0188] Among them, wash buffer: 8M urea, 100mM NaH2PO4, 10mM Tris, 20mM imidazole, and the rest is water.

[0189] Elute buffer: 8M urea, 100mM NaH2PO4, 10mM Tris, 250mM imidazole, the balance is water.

[0190] 2. In vitro droplet analysis

[0191] In the buffer (25mM Tris-HCl pH7.5), in vitro purified 6×His-SGF29 was added, and the concentration of the protein in the system was 5μM. The resulting system was subjected to an in vitro phase separation experiment at room temperature, and BSA was used to replace the protein as a negative control. The phase-separated protein solution was then loaded onto a slide (microscope glass slide), and the slide was imaged using an Andor confocal microscope with a ×150 objective lens, or the absorbance value was measured using an ELISA reader.

[0192] Results Figure 3 In Figure A, the results of phase separation of 5 μM in vitro purified 6×His-SGF29 protein showed that the negative control BSA did not form a turbid solution in the buffer, while the in vitro purified 6×His-SGF29 protein could form a turbid solution. Similarly, when using an ELISA instrument to measure the absorbance value, it can be observed that the absorbance value of the 6×His-SGF29 solution that underwent phase separation in vitro was significantly higher than that of the negative control BSA solution, indicating that the in vitro purified 6×His-SGF29 has the ability to undergo phase separation in vitro.

[0193] After the above phase separation experiment, the system was centrifuged at 13,000×g for 10 min, the supernatant was collected, and the precipitate was resuspended with 1×SDS loading buffer (Dingguo Changsheng, WB-0091). The resuspension and the supernatant were subjected to polyacrylamide gel electrophoresis, and the liquid before centrifugation was used as a control.

[0194] Results Figure 3 In Figure B, the results show that the negative control BSA protein solution did not undergo phase separation and could not be precipitated after centrifugation. In contrast, the 6×His-SGF29 protein was enriched in the precipitate after centrifugation due to phase separation, further indicating that the in vitro purified 6×His-SGF29 underwent phase separation.

[0195] Example 4: Mutation of SGF29 can disrupt phase separation caused by SGF29

[0196] 1. Disordered and irregular scoring

[0197] The FASTA sequence of SGF29 protein (NP_612423.1, updated on May 17, 2023) was input into http: / / www.pondr.com / , and VLXT was selected for disordered and irregular scoring.

[0198] result( Figure 4 Middle A) shows that there are a large number of disordered and irregular sequences near the 207th amino acid of SGF29.

[0199] 2. Western blot detection

[0200] Transfection was performed according to the method of step 3 of Example 2. The cells used were hMPCs of passage P5. The viruses used were the EGFP-SGF29 lentivirus and the EGFP-SGF29 R207P lentivirus of step 1 of Example 2.

[0201] The protein levels of SGF29 R207P expressed in human mesenchymal stem cells were analyzed by Western blotting, using human mesenchymal stem cells as a control. Figure 4 Middle B) shows that the expression levels of EGFP-SGF29 (i.e. WT) and SGF29 R207P in human mesenchymal stem cells are relatively close. The antibodies used are SGF29 (SantaCruz, sc-515286) and GAPDH antibody (SantaCruz, sc-25778).

[0202] 3. Immunofluorescence

[0203] Transfection was performed according to the method of step 3 of Example 2. The cells used were hMPCs of passage P5. The viruses used were the EGFP-SGF29 lentivirus and the EGFP-SGF29 R207P lentivirus of step 1 of Example 2.

[0204] EGFP-SGF29 (WT) or EGFP-SGF29 R207P was expressed in mesenchymal stem cells, and the ability of SGF29 and SGF29 R207P to undergo phase transition was observed by immunofluorescence. The primary antibody used was Lamin A / C antibody (E-1) (Santa, sc-376248), and the secondary antibody was Goat anti-Rabbit IgG1 Cross-Adsorbed SecondaryAntibody, Alexa Fluor 568 (Invitrogen, A11057).

[0205] result( Figure 4 Figures C and D show that only SGF29 undergoes phase separation, while the R207P mutant (EGFP-SGF29 R207P) cannot undergo phase separation well. Statistical results show that the number of SGF29 phase transition droplets in each cell increases after EGFP-SGF29 is expressed in mesenchymal stem cells, further indicating that EGFP-SGF29 has the characteristics of phase separation.

[0206] 4. Fluorescence photobleaching recovery experiment

[0207] Transfection was performed according to the method of step 3 of Example 2. The cells used were young human mesenchymal stem cells (P5_hMPCs); the viruses used were the EGFP-SGF29 lentivirus and the EGFP-SGF29 R207P lentivirus of step 1 of Example 2.

[0208] According to step 6 of Example 2, a fluorescence recovery after photobleaching (FRAP) experiment was performed. The results were ( Figure 4 Middle E) shows that wild-type EGFP-SGF29 (WT) recovers more slowly than EGFP-SGF29 R207P, further indicating that EGFP-SGF29 undergoes phase separation.

[0209] Example 5: Mutation of SGF29 can restore cell senescence caused by SGF29

[0210] 1. SA-β-Gal staining experiment

[0211] Transfection was performed according to the method of step 3 of Example 2. The cells used were young human mesenchymal stem cells (P5_hMPCs); the viruses used were the EGFP lentivirus, EGFP-SGF29 lentivirus and EGFP-SGF29 R207P lentivirus of step 1 of Example 2.

[0212] According to the method of step 3 of Example 1, the cells after virus transfection were subjected to SA-β-gal staining experiment. The results were ( Figure 5 Middle A) shows that the expression of EGFP-SGF29 (WT) can promote the aging of human mesenchymal stem cells, indicating that the expression of SGF29 can promote the aging of human mesenchymal stem cells; the aging of cells expressing EGFP-SGF29 R207P is not significantly different from that of the control (cells expressing EGFP), indicating that the cell senescence caused by SGF29 can be restored after mutating SGF29 to SGF29 R207P.

[0213] 2. Monoclonal formation ability experiment

[0214] Transfection was performed according to the method of step 3 of Example 2. The cells used were young human mesenchymal stem cells (P5_hMPCs); the viruses used were the EGFP lentivirus, EGFP-SGF29 lentivirus and EGFP-SGF29 R207P lentivirus of step 1 of Example 2.

[0215] According to the method of step 4 of Example 1, the cells after virus transfection were subjected to a monoclonal formation ability test. The results were ( Figure 5Middle B) shows that the expression of EGFP-SGF29T can significantly inhibit the monoclonal formation ability of young human mesenchymal stem cells; the monoclonal formation ability of cells expressing EGFP-SGF29 R207P is not significantly different from that of the control (cells expressing EGFP), indicating that the monoclonal formation ability of cells can be restored after SGF29 is mutated to SGF29 R207P.

[0216] 3. Immunofluorescence experiment

[0217] Transfection was performed according to the method of step 3 of Example 2. The cells used were young human mesenchymal stem cells (P5_hMPCs); the viruses used were the EGFP lentivirus, EGFP-SGF29 lentivirus and EGFP-SGF29 R207P lentivirus of step 1 of Example 2.

[0218] According to the method of step 6 of Example 1, Ki67 immunofluorescence experiment was performed on the virus-transfected cells. The antibody used was Ki67 antibody (ZSGB-BIO, ZM-0166,). The results were ( Figure 5 Middle C) shows that the expression of EGFP-SGF29 (WT) promotes the proportion of Ki67-positive cells in human mesenchymal stem cells, while the proportion of Ki67-positive cells expressing EGFP-SGF29 R207P is not significantly different from the control (cells expressing EGFP), further indicating that the expression of EGFP-SGF29 can promote the aging of human mesenchymal stem cells, the aging of cells can be restored after SGF29 is mutated to SGF29 R207P, and the phase separation of EGFP-SGF9 can promote the aging of human mesenchymal stem cells.

[0219] Example 6: SGF29 can promote the expression of CDKN1A

[0220] 1. ChIP-qPCR

[0221] Transfection was performed according to the method of step three of Example 2. The cells used were young human mesenchymal stem cells (P5_hMPCs); the viruses used were the EGFP lentivirus (with a Flag tag, also recorded as Flag-EGFP lentivirus), EGFP-SGF29 lentivirus (with a Flag tag, also recorded as Flag-EGFP-SGF29 lentivirus) and EGFP-SGF29R207P lentivirus (with a Flag tag, also recorded as Flag-EGFP-SGF29 R207P lentivirus) of step one of Example 2.

[0222] Collect 5×10 6After infection, SGF29 ChIP-qPCR was performed to detect the relative enrichment of CDKN1A using Flag antibody (Invitrogen, F1804), and ChIP-qPCR was performed on cells expressing EGFP using IgG. Results ( Figure 6 Middle A) shows that EGFP-SGF29 promotes the enrichment of SGF29 on CDKN1A, while the mutation of SGF29 inhibits the enrichment of SGF29 on CDKN1A, indicating that overexpression of SGF29 can promote the expression of CDKN1A, while the mutation of SGF29 can inhibit the promotion of SGF29 on CDKN1A expression. The primers used for qPCR are as follows:

[0223] CDKN1A promoter ChIP-qPCR Forward:CCAGCCCTTGGATGTGTTT;

[0224] CDKN1A promoter ChIP-qPCR Reverse:GCTCCTTTCTGTGCCTGA.

[0225] 2. DNA FISH

[0226] Transfection was performed according to the method of step 3 of Example 2. The cells used were young human mesenchymal stem cells (P5_hMPCs); the viruses used were the Flag-EGFP lentivirus, Flag-EGFP-SGF29 lentivirus and Flag-EGFP-SGF29 R207P lentivirus of step 1 of Example 2.

[0227] The probes used are listed in Table 1.

[0228] Table 1. Probe sequences

[0229]

[0230]

[0231]

[0232]

[0233]

[0234]

[0235]

[0236]

[0237] The results are as follows Figure 6 As shown in B, the results showed that the SGF29 dot-like structure and the two red CDKN1A promoter signal points co-localized, indicating that SGF29 formed a dot-like structure on the CDKN1A gene promoter.

[0238] 3. qPCR

[0239] Transfection was performed according to the method of step 3 of Example 2. The cells used were young mesenchymal stem cells (P5_hMPCs); the viruses used were the EGFP lentivirus, EGFP-SGF29 lentivirus, and EGFP-SGF29 R207P lentivirus in step 1 of Example 2. The CDKN1A gene (encoding p21 Cip1 The primers used were CDKN1A promoter ChIP-qPCR Forward and CDKN1A promoter ChIP-qPCR Reverse as described above.

[0240] CDKN1A promoter ChIP-qPCR:Forward CCAGCCCTTGGATGTGTTT;

[0241] CDKN1A promoter ChIP-qPCR:Reverse GCCTCCTTTCTGTGCCTGA.

[0242] The results are as follows Figure 6 As shown in C, the results show that the expression of SGF29 can promote the expression of CDKN1A, while the mutation of SGF29 can inhibit the promotion of CDKN1A expression by SGF29, indicating that the expression of SGF29 promotes the aging of human mesenchymal stem cells by promoting the mRNA expression of CDKN1A.

[0243] 4. Western blot detection

[0244] Transfection was performed according to the method of step 3 of Example 2. The cells used were young mesenchymal stem cells (P5_hMPCs); the viruses used were the EGFP lentivirus, EGFP-SGF29 lentivirus, and EGFP-SGF29 R207P lentivirus of step 1 of Example 2. The infected young mesenchymal stem cells were taken and p21 was detected by Western blotting. Cip1 The internal control used was β-Tubulin. The antibody used was P21 Cip1 Antibody (Cell signaling Technology, CST2974s) and Tubulin antibody.

[0245] The results are as follows Figure 6 As shown in D, the results show that EGFP-SGF29 can indeed promote the expression of p21 in young human mesenchymal stem cells. Cip1 The protein expression level of SGF29 was inhibited by SGF29 mutation. Cip1 The promotion of expression indicates that SGF29 expression promotes the aging of human mesenchymal stem cells because it promotes the mRNA expression of CDKN1A.

[0246] 5. siRNA interference experiment

[0247] 1. Transfection was performed according to the method of step 3 of Example 2. The cells used were young mesenchymal stem cells (P5_hMPCs); the viruses used were the EGFP lentivirus and EGFP-SGF29 lentivirus in step 1 of Example 2. The infected young mesenchymal stem cells were subcultured for 5 generations.

[0248] 2. After step 1, inoculate into 6-well plates, 1×10 5 cells.

[0249] 3. Add 20 pmol of siRNA to 250 μl of Opti-MEM serum-free medium and mix gently to obtain diluted siRNA. The siRNAs used are si-Control, si-CDKN1A#1, and si-CDKN1A#2. The sequences of each siRNA are as follows:

[0250] si-Control:UUCUCCGAACGUGUCACGU;

[0251] si-CDKN1A#1: GAUGGAACUUCGACUUUGU; si-CDKN1A#2: CUGAUCUUCUCCAAGAGGA.

[0252] 4. Mix lipofectamin reagent: dilute 1 μl lipofectamin reagent with 250 μl Opti-MEM serum-free medium, mix gently and place at room temperature for 5 minutes:

[0253] 5. Mix the diluted siRNA in step 3 and the Lipofectamin reagent in step 4 gently and leave at room temperature for 20 minutes to form a siRNA / lipofectamin complex.

[0254] 6. Add 100 μl of siRNA / lipofectamin complex to the wells of the culture plate containing cells and culture medium in step 2, and gently shake the cell culture plate back and forth to perform siRNA interference.

[0255] Western blot was performed 48 hours after siRNA interference, and young mesenchymal stem cells infected with EGFP lentivirus and EGFP-SGF29 lentivirus without siRNA interference were used as controls. Cip1 Antibodies (Cellsignaling Technology, CST2974s), GFP (Novus Biologicals, NB120-6660). Results are shown in Figure 6 Middle E.

[0256] The young mesenchymal stem cells were subjected to SA-β-gal staining experiment after 48 hours of siRNA interference. Figure 6 Middle F.

[0257] The results showed that siRNA could indeed inhibit the expression of CDKN1A and significantly reduce the expression of p21 induced by SGF29. Cip1 Increased levels of the protein cause cellular senescence.

[0258] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. Use of substances that detect phase separation in cells in the preparation of products for detecting or assisting in the detection of aging, or in the detection or assisting in the detection of aging.

2. Use of substances that detect phase separation in cells in the preparation of products for screening or assisting in the screening of senescent cells, or in the screening or assisting in the screening of senescent cells.

3. The use according to claim 1 or 2, Features: The phase separation is caused by SGF29.

4. Use of a substance for detecting the amount of SGF29 gene expression in cells, or a substance for detecting the content of SGF29 in cells, in the preparation of products for detecting or assisting in the detection of aging, or in the detection or assisting in the detection of aging.

5. The use according to any one of claims 1 to 4, Features: The aging refers to the aging of animal cells, tissues, organs or individuals.

6. Methods for preparing senescent cell models, include: Senescent cells are obtained by increasing the content or activity of SGF29 in the receptor cells, or promoting the expression of the SGF29 gene in the receptor cells, or promoting the phase separation of SGF29 in the receptor cells.

7. The method according to claim 6, Features: The method is achieved by introducing the SGF29 gene into the recipient cell and expressing the SGF29 gene.

8. Use of a substance that inhibits SGF29 gene expression or knocks out SGF29 gene in the preparation of a product for treating and / or preventing and / or delaying aging, or in treating and / or preventing and / or delaying aging.

9. Use of a substance that reduces the content or activity of SGF29 in the preparation of a product for treating and / or preventing and / or delaying aging, or in treating and / or preventing and / or delaying aging.

10. Use of a substance that inhibits phase separation caused by SGF29 in cells in the preparation of a product for treating and / or preventing and / or delaying aging, or in treating and / or preventing and / or delaying aging.

11. The use according to any one of claims 8 to 10, Features: The aging refers to the aging of animal cells, tissues, organs or individuals.

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