Construction method and application of cell senescence model

Abstract

The invention discloses a construction method and application of a cell senescence model, and belongs to the technical field of biology, firstly, cells are cultured in an embryonic-stage animal serum complete medium to a logarithmic phase for passage, then the cells are inoculated with an embryonic-stage animal serum complete medium, a newborn-stage animal serum complete medium or an adult-stage animal serum complete medium respectively, and the cell senescence model is obtained. The method comprises the following steps of: culturing and observing the cell state, and determining cell senescence by SA-beta-gal staining and detecting the transcriptional level of senescence-related genes when the cells grow slowly and become large and flat in comparison with an animal serum group in an embryonic period, so as to prove that the cell senescence model is successfully constructed. The cell senescence model is simple and convenient to operate, and the senescence induction process is simple, stable and rapid; the cell senescence model induced by newborn animal serum and adult animal serum simulates the senescence process of organisms of different age groups in vitro, and breaks through the limitation that cell senescence phenotypes are generated by oxidative stress treatment.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a method for constructing a cell aging model and an application thereof. Background Art

[0002] When cells lose their ability to replicate and enter an irreversible state of proliferation arrest, it is called cell senescence, also known as replicative senescence (RS). Cell senescence is a response to stress and damage and increases with age. Senescent cells enter a state of permanent growth arrest and release a senescence-associated secretory phenotype. Cell senescence has beneficial biological functions in regulating embryonic development, wound healing, fibrosis regression, and tumor suppression. However, long-term senescence may lead to harmful sequelae, including tumor development, chronic inflammation, immune deficiency, and stem cell exhaustion. Due to the observation that cell senescence plays an important role in the pathogenesis of chronic diseases such as osteoporosis, metabolic syndrome, type 2 diabetes, cancer, reproductive senescence, atherosclerosis, neurodegenerative diseases, glaucoma, and chronic kidney disease, people are increasingly interested in cell senescence and interventions to regulate senescence.

[0003] There are currently two known ways to induce cell senescence: replicative senescence and stress-induced premature senescence. In the first case, cell senescence is caused by telomere attrition after repeated proliferation; in the second case, cell senescence is caused by subtoxic doses of external or intrinsic stimuli, such as radiation, oxidative stress, drugs, and carcinogenic stress (doi:10.1016 / j.cell.2019.10.005). However, the first method of constructing a cell senescence model is mainly used for primary cells and is time-consuming. The second method, such as oxidative stress treatment, will produce a short-term aging phenotype, causing cells to rapidly apoptosis and die within a few days, and it is easy to induce cell apoptosis rather than cell senescence. A single type of senescent cell model can often only simulate some of the characteristics of the aging process. For example, H2O2-treated cells mainly simulate DNA damage caused by oxidative stress during aging, but cannot reflect the entire process of cell aging. Summary of the invention

[0004] In view of the above problems, the primary purpose of the present invention is to provide a method for constructing a cell aging model that is simple, stable, rapid, and capable of reflecting the aging process. Specifically, it involves replacing embryonic animal serum in the complete cell culture medium with neonatal animal serum or adult animal serum, and culturing cells under this system for 3-10 days to induce cell aging.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] The following three groups of culture media were prepared: basal culture medium + embryonic animal serum, basal culture medium + neonatal animal serum, and basal culture medium + adult animal serum. The first group was the control group, and the second and third groups were modeling groups. The cells were cultured in complete culture medium with embryonic animal serum until the logarithmic growth phase and then passaged. The culture medium components were replaced according to the above three experimental groups before inoculating the cells. After culturing the cells for 3-10 days, the cell status was observed. Compared with the embryonic animal serum group, the cells in the neonatal animal serum and adult animal serum groups grew slower, and the cells became larger and flatter. Cell senescence was determined by detecting aging-related genes using SA-β-gal staining and qPCR.

[0007] The cells are derived from human or mouse. The human cells are human mesenchymal stem cells and human immortalized keratinocytes, and the mouse cells are mouse myoblasts and mouse embryonic osteoblasts.

[0008] The cells are selected from mouse myoblasts, and the culture medium is selected from DMEM, double antibodies, and newborn animal serum, or DMEM, double antibodies, and adult animal serum.

[0009] The cells are selected from mouse embryonic osteoblasts, and the culture medium is selected from MEMα, double antibody, and newborn animal serum, or MEMα, double antibody, and adult animal serum.

[0010] The cells are human mesenchymal stem cells, and the culture medium is DMEM, double antibody, L-glutamine, and newborn animal serum, or DMEM, double antibody, L-glutamine, and adult animal serum.

[0011] The cells are human immortalized keratinocytes, and the culture medium is DMEM, double antibodies, and newborn animal serum, or DMEM, double antibodies, and adult animal serum.

[0012] The present invention also provides a cell aging model, which is constructed using the above-mentioned construction method.

[0013] The present invention also proposes the application of the above-mentioned cell aging model in the screening of drugs for alleviating and / or preventing cell aging.

[0014] The advantages of the cell senescence model induced by neonatal animal serum or adult animal serum constructed by the method of the present invention are: (1) compared with operations such as radiation and oxidative stress on cells, the cell senescence model is easy to operate, neonatal animal serum and adult animal serum are easy to obtain, and the process of inducing cell senescence by neonatal animal serum and adult animal serum is similar to the method of culturing cells in complete cell culture medium, and the process is simple; (2) The cell senescence model induced by neonatal animal serum and adult animal serum simulates the aging process of the body at different ages in vitro, breaking through the limitation of oxidative stress treatment in producing cell senescence phenotype. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The effect of fetal bovine serum (FBS), newborn calf serum (NCS) and adult bovine serum (ABS) culture on the aging phenotype of mouse myoblasts (C2C12) in the embodiment of the present invention. Among them, (a) is the SA-β-gal staining result, showing that on the sixth day of cell culture, both the NCS group and the ABS group showed aging phenomenon, with obvious positive staining cells, and the FBS group had no obvious aging phenomenon. (b) is the qPCR identification of the expression of aging-related genes. Compared with the FBS group, the expression of the aging-upregulated gene Cdkn1a in the NCS group and the ABS group increased, the expression of the aging-related secretion phenotype interleukin Il6 and matrix metalloproteinase Mmp3 increased, and the expression of the aging-downregulated gene Lmnb1 decreased. (c) is the growth curve. As the culture time increased, starting from the fourth day, the cell proliferation rate in the NCS group and the ABS group slowed down significantly. (ns, no significant, *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001).

[0016] Figure 2 The effect of fetal bovine serum (FBS), newborn calf serum (NCS) and adult bovine serum (ABS) culture on the aging phenotype of mouse embryonic osteoblasts (MC3T3-E1) in the embodiment of the present invention. Among them, (a) is the SA-β-gal staining result, showing that on the sixth day of cell culture, both the NCS and ABS groups showed aging phenomena, with obvious positive staining cells, and the FBS group had no obvious aging phenomenon. (b) is the qPCR identification of the expression of aging-related genes. Compared with the FBS group, the expression of the aging-upregulated gene Cdkn1a in the NCS group and the ABS group increased, the expression of the aging-related secretion phenotype interleukin Il6 increased, and the expression of the aging-downregulated gene Lmnb1 decreased. (c) is the growth curve. As the culture time increased, starting from the fourth day, the cell proliferation rate in the NCS group and the ABS group slowed down significantly. (ns, no significant, *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001).

[0017] Figure 3 The effects of fetal bovine serum (FBS), newborn calf serum (NCS) and adult bovine serum (ABS) culture on the aging phenotype of human mesenchymal stem cells (hMSC) and human immortalized keratinocytes (HaCaT) in the embodiments of the present invention. (a) SA-β-gal staining results show that on the sixth day of human mesenchymal stem cell culture, both the NCS group and the ABS group showed aging phenomena, with obvious positive staining cells, while the FBS group had no obvious aging phenomenon. (b) SA-β-gal staining results show that on the sixth day of human immortalized keratinocyte culture, both the NCS group and the ABS group showed aging phenomena, with obvious positive staining cells, while the FBS group had no obvious aging phenomenon. (ns, no significant, *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001). DETAILED DESCRIPTION

[0018] The present invention is further described in detail below in conjunction with specific embodiments. The examples given are only for illustrating the present invention, not for limiting the scope of the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The materials, reagents, instruments, etc. used in the following examples are all commercially available unless otherwise specified. The quantitative tests in the following examples were repeated three times, and the results were averaged. The cell culture conditions in the following examples were all 37 degrees Celsius, 5% CO2 unless otherwise specified.

[0019] Newborn calf serum: Vivacell;

[0020] Adult bovine serum: Vivacell;

[0021] Fetal bovine serum: Sangon Biotech.

[0022] Example 1: Establishment of a mouse myoblast (C2C12) aging model induced by newborn calf serum or adult calf serum

[0023] After the cells recovered and reached a certain number, they were passaged once and then cultured at 1.5×10 4The cells were passaged into 12-well cell culture plates, and the experimental groups were set as DMEM (Dulbecco's Modified Eagle Medium) + 1% Penicillin-Streptomycin Solution (P / S, penicillin-streptomycin solution) + 10% fetal bovine serum (44.5 mL DMEM + 500 μL P / S + 5 mL fetal bovine serum), DMEM + 1% P / S + 10% newborn bovine serum (44.5 mL DMEM + 500 μL P / S + 5 mL newborn bovine serum), and DMEM + 1% P / S + 10% adult bovine serum (44.5 mL DMEM + 500 μL P / S + 5 mL adult bovine serum). The culture components were replaced according to the experimental groups before inoculating the cells. The cell status was observed until the sixth day, and the cell senescence level was assessed by SA-β-gal staining. RNA samples were collected to detect the transcription levels of senescence-related genes Cdkn2a, Cdkn1a, Mmp3, Il6 and Lmnb1, and all were compared with DMEM+1% P / S+10% fetal bovine serum group cells for control experiments. Cell growth curves were measured, and the number of cells in the three groups was recorded after complete attachment (i.e. Day0), 24h after attachment (i.e. Day1), 48h after attachment (i.e. Day2), Day3, Day4, Day5 and until Day6, and the cell growth curve was drawn.

[0024] The experimental results showed that compared with the cells in the fetal bovine serum group, the newborn bovine serum group and the adult bovine serum group showed aging phenomena, with obvious positive staining cells. Figure 1 (a) In the newborn bovine serum group and the adult bovine serum group, the expression of the aging-upregulated gene Cdkn1a increased, the expression of the aging-related secretory phenotypes Il6 and Mmp3 increased, and the expression of the aging-downregulated gene Lmnb1 decreased. Figure 1 (b) The growth curves of cells in three culture systems, namely, fetal bovine serum, newborn calf serum and adult bovine serum, show that with the increase of culture time, starting from the fourth day, the cell proliferation rate in the newborn calf serum group and the adult bovine serum group slowed down. Figure 1 (c).

[0025] Example 2: Establishment of an aging model of mouse embryonic osteoblasts (MC3T3-E1) induced by newborn calf serum or adult calf serum

[0026] After the cells recovered and reached a certain number, they were passaged once and then cultured at 1.5×10 4The cells were passaged into 12-well cell culture plates, and the experimental groups were set as MEMα+1%P / S+10% fetal bovine serum (44.5mL MEMα+500μL P / S+5mL fetal bovine serum), MEMα+1%P / S+10% newborn bovine serum (44.5mL MEMα+500μL P / S+5mL newborn bovine serum), and MEMα+1%P / S+10% adult bovine serum (44.5mL MEMα+500μL P / S+5mL adult bovine serum). The culture components were replaced according to the experimental groups before inoculating the cells. The cell state was observed until the sixth day, and the cell senescence level was evaluated by SA-β-gal staining. RNA samples were collected to detect the transcription levels of senescence-related genes Cdkn2a, Cdkn1a, Mmp3, Il6 and Lmnb1, and all were compared with cells in the MEMα+1% P / S+10% fetal bovine serum group for control experiments. Cell growth curves were measured, and the number of cells in the three groups was recorded after complete attachment (i.e. Day0), 24h after attachment (i.e. Day1), 48h after attachment (i.e. Day2), Day3, Day4, Day5 and until Day6, and the cell growth curve was drawn.

[0027] The experimental results showed that compared with the cells in the fetal bovine serum group, the newborn bovine serum group and the adult bovine serum group showed aging phenomena, with obvious positive staining cells. Figure 2 (a) In the newborn bovine serum group and the adult bovine serum group, the expression of the aging-upregulated gene Cdkn1a increased, the expression of the aging-related secretory phenotype Il6 increased, and the expression of the aging-downregulated gene Lmnb1 decreased. Figure 2 (b) The growth curves of cells in three culture systems, namely, fetal bovine serum, newborn calf serum and adult bovine serum, show that with the increase of culture time, starting from the fourth day, the cell proliferation rate in the newborn calf serum group and the adult bovine serum group slowed down. Figure 2 (c).

[0028] Example 3: Establishment of a human mesenchymal stem cell (hMSC) aging model induced by newborn calf serum or adult calf serum

[0029] After the cells recovered and reached a certain number, they were passaged once and then cultured at 1.5×10 4The cells were passaged into 12-well cell culture plates, and the experimental groups were set as DMEM+1%P / S+2mM L-glutamine+10% fetal bovine serum (44mL DMEM+500μL P / S+500μL 200mM L-glutamine+5mL fetal bovine serum), DMEM+1%P / S+2mM L-glutamine+10% newborn bovine serum (44mL DMEM+500μL P / S+500μL 200mM L-glutamine+5mL newborn bovine serum), and DMEM+1%P / S+2mM L-glutamine+10% adult bovine serum (44mL DMEM+500μL P / S+500μL200mM L-glutamine+5mL adult bovine serum). The culture components were replaced according to the experimental groups before inoculation of cells. The cell status was observed until the sixth day, and the cell senescence level was assessed by SA-β-gal staining.

[0030] The experimental results showed that compared with the cells in the fetal bovine serum group, the newborn bovine serum group and the adult bovine serum group showed aging phenomena, with obvious positive staining cells. Figure 3 (a).

[0031] Example 4: Establishment of a human immortalized keratinocyte (HaCaT) aging model induced by newborn calf serum or adult calf serum

[0032] After the cells recovered and reached a certain number, they were passaged once and then cultured at 1.5×10 4 The cells were passaged into 12-well cell culture plates, and the experimental groups were set as DMEM+1%P / S+2% fetal bovine serum (48.5mL DMEM+500μL P / S+1mL fetal bovine serum), DMEM+1%P / S+2% newborn bovine serum (48.5mL DMEM+500μL P / S+1mL newborn bovine serum), and DMEM+1%P / S+2% adult bovine serum (48.5mL DMEM+500μL P / S+1mL adult bovine serum). The culture components were replaced according to the experimental groups before inoculating the cells. The cell status was observed until the sixth day, and the cell senescence level was assessed by SA-β-gal staining.

[0033] The experimental results showed that compared with the cells in the fetal bovine serum group, the cells in the newborn bovine serum group and the adult bovine serum group showed senescence, with obvious positive staining cells. Figure 3 (b).

Claims

1. A method for constructing a cell aging model, characterized in that: First, the cells were cultured in complete culture medium with embryonic animal serum and then passaged when they reached the logarithmic growth phase. Then, the cells were inoculated with complete culture medium with embryonic animal serum, neonatal animal serum, or adult animal serum, respectively, and cultured to observe the cell status. Compared with the embryonic animal serum group, the cells in the neonatal animal serum or adult animal serum group grew slower, and the cells became larger and flatter. Cell senescence was determined by SA-β-gal staining and detection of transcription levels of aging-related genes, demonstrating that the cell senescence model was successfully constructed.

2. The method for constructing a cell aging model according to claim 1, characterized in that: The cell culture time is 3-10 days.

3. The method for constructing a cell aging model according to claim 1, characterized in that: The cells are derived from human or mouse. The human cells are human mesenchymal stem cells and human immortalized keratinocytes, and the mouse cells are mouse myoblasts and mouse embryonic osteoblasts.

4. The method for constructing a cell aging model according to claim 3, characterized in that: The cells are selected from mouse myoblasts, and the complete culture medium is selected from DMEM, double antibodies, and newborn animal serum, or DMEM, double antibodies, and adult animal serum.

5. The method for constructing a cell aging model according to claim 3, characterized in that: The cells are selected from mouse embryonic osteoblasts, and the complete culture medium is selected from MEMα, double antibody, and newborn animal serum, or MEMα, double antibody, and adult animal serum.

6. The method for constructing a cell aging model according to claim 3, characterized in that: The cells are human mesenchymal stem cells, and the complete culture medium is DMEM, double antibody, L-glutamine, and newborn animal serum, or DMEM, double antibody, L-glutamine, and adult animal serum.

7. The method for constructing a cell aging model according to claim 3, characterized in that: The cells are human immortalized keratinocytes, and the complete culture medium is DMEM, double antibodies, and newborn animal serum, or DMEM, double antibodies, and adult animal serum.

8. A cell aging model, characterized in that: The method is constructed by any one of claims 1 to 7.

9. Use of the cell senescence model according to claim 8 in screening drugs for alleviating and / or preventing cell senescence.

Images