Establishment method and application of red deer pilose antler cartilage primary cell senescence model

Abstract

The invention provides an establishment method and application of a red deer pilose antler cartilage primary cell senescence model, and belongs to the technical field of biology. According to the invention, a hydrogen peroxide (H2O2) induction mode is used, and a novel cell aging model of red deer antler cartilage primary cells is constructed. And determining that the aging half effect concentration of H2O2 as an inducer of the cells is 400 mu M, and the induction time is 2 hours. The cell senescence model provided by the invention is simple to operate, low in cost, short in cell induction time, stable in result and high in repeatability, and experimenters can quickly and effectively construct the cell senescence model. Importantly, the cell aging model in the regeneration background can simulate the oxidative stress state in the aging process of a specific regeneration organ pilose antler, can be used for screening and research and development of small molecular compounds for preventing and delaying aging, and can deeply compare the mechanisms and functions of regeneration and aging at the cellular level; and an important cell platform is provided for better understanding the relationship between regeneration and aging.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a method for establishing a primary cell aging model of red deer antler cartilage and application thereof. Background Art

[0002] With the advancement of medical technology and the increasing problem of population aging, people attach great importance to the study of aging. Cellular senescence refers to a stable cell cycle arrest state, when proliferating cells become tolerant to growth-promoting stimuli, which are usually caused by DNA damage. Cellular senescence usually exhibits the following characteristics: morphological and metabolic changes, chromatin remodeling, changes in gene expression, and the appearance of senescence-associated secretory phenotype (SASP).

[0003] At present, among several types of cell aging models, such as replicative, inducible and oncogene-induced, the induced aging model is widely used because of its short modeling time and high induction success rate. As a small molecule strong oxidant, hydrogen peroxide (H2O2) is easily available and is the most commonly used aging inducer in the construction of induced aging models. Endogenous H2O2 products are also produced in the cell's own metabolic process, which is a signal molecule for some cell activities. As an inducer, H2O2 is easy to enter the cell. Its strong oxidative properties cause the cell to produce a large amount of reactive oxygen species (ROS) in a short period of time, leading to intracellular DNA damage. The DNA damage signal activates the stress sensor protein ATM / ATR and causes a tumor suppressor protein TP53 to be phosphorylated. The activated TP53 promotes the expression of downstream cell cycle regulatory genes such as the cyclin-dependent kinase inhibitor P21, triggering cell cycle arrest and causing the cell to enter a senescent state.

[0004] Regeneration is a physiological process that has the opposite function of aging. It restores damaged tissues and replaces aging cells. However, regeneration, especially regeneration at the organ / appendage level, is rare in mammals. Deer antler is the only organ in adult mammals known to be able to fully regenerate periodically. It is not only an ideal model for studying regeneration mechanisms, but also has high value for revealing aging mechanisms. However, there is no such cellular aging model.

[0005] Rapid antler regeneration includes two processes: chondrogenesis and osteogenesis. Chondrogenesis occurs in the antler tip growth zone near the skull. During this process, the cells at the antler tip proliferation point continue to grow, develop, and differentiate into cartilage tissue (CA). Constructing a cell aging model in a regeneration context can more deeply compare the mechanisms and functions of regeneration and aging at the cellular level, providing important support for understanding the relationship between regeneration and aging. Therefore, it is particularly important to construct a cell aging model in such a context. Summary of the invention

[0006] In view of the above situation, the present invention uses hydrogen peroxide (H2O2) to induce primary antler cells, proposes a method for establishing an aging model of primary cells of red deer antler cartilage and its application, so as to provide a cell aging model in a regeneration background, provide a cell platform for studying the relationship between regeneration and aging, and apply it in the screening of small molecule compounds that delay cell aging.

[0007] One of the purposes of the present invention is to provide a method for establishing an aging model based on primary cells of red deer antler cartilage, using hydrogen peroxide (H2O2) to induce primary cells of red deer antler cartilage to construct a cell aging model. The second purpose of the present invention is to determine that the aging half-effective concentration of H2O2, an inducer of primary cells of red deer antler cartilage, is 400 μM, and the induction time is 2 hours. The present invention uses CCK-8 method for screening, and combines β-galactosidase (SA-β-GAL) staining to analyze cell aging to determine the H2O2 modeling concentration and induction time. The third purpose of the present invention is to use real-time quantitative PCR (RT-qPCR) and Western Blotting methods to detect the changes in transcription and protein expression of key marker genes of the TP53-CDKN1A (P21) axis that controls aging in cells to determine the success of modeling. The fourth purpose of the present invention is the application of the established cell aging model in the screening of small molecule compounds that delay cell aging.

[0008] The present invention is implemented by the following technical solutions:

[0009] 100-1600 μM H2O2 was added to the complete culture medium to induce the primary cells of antler cartilage for 0.5-2 h. The culture medium was replaced with complete culture medium and cultured for 72 h. The CCK-8 method was used to analyze the aging status in combination with SA-β-GAL staining to determine that the half-effective concentration for modeling was 400 μM and the induction time was 2 h. Significant changes in the expression levels of cell aging marker genes were obtained.

[0010] Furthermore, the primary cells are derived from antler cartilage tissue of red deer in a rapid growth period.

[0011] Furthermore, the complete culture medium components include: DMEM, 10% fetal bovine serum, 1% penicillin / streptomycin, and the serum-free culture medium components include: DMEM, 1% penicillin / streptomycin.

[0012] Furthermore, the CCK-8 method was used in combination with SA-β-GAL staining to analyze cell senescence, and the half-effective concentration of the modeled inducer H2O2 was determined to be 400 μM, and the induction time was 2 h.

[0013] Furthermore, RT-qPCR and Western Blotting methods were used to detect changes in transcription and protein expression of key marker genes of the TP53-CDKN1A (P21) axis that controls aging in cells to confirm the success of the modeling.

[0014] Compared with the existing cell models, the beneficial effects of the present invention are:

[0015] (1) The method provided by the present invention is simple to operate, low in cost, has a short cell induction time, stable results, and strong repeatability, which helps experimenters to quickly and effectively construct a cell aging model with regenerative function.

[0016] (2) Constructing a cell aging model in a regenerative context can enable in-depth comparison of mechanisms and functions at the cellular level, providing an important cellular platform for understanding the relationship between regeneration and aging. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Effects of different concentrations of H2O2 on the survival rate of primary antler cartilage cells (P<0.001);

[0018] Figure 2 Results of SA-β-GAL staining of primary antler cartilage cells induced by different concentrations of H2O2. The left pictures in the four groups A, B, C, and D are the morphology of unstained cells in the control group (10×10); the right pictures in the four groups A, B, C, and D are the morphology of cells after staining in the experimental group (10×10).

[0019] Figure 3 The positive staining rate of SA-β-GAL stained cells after senescence of primary antler cartilage cells induced by different concentrations of H2O2 (P<0.001);

[0020] Figure 4 Comparison of TP53 (A) and P21 (B) gene transcriptional expression abundance in the aging model control group (NC) and experimental group of primary antler cartilage cells after H2O2 treatment for 2h (P<0.001). NC: control group; H2O2 treated (400μM): experimental group.

[0021] Figure 5Comparison of TP53 (A, B) and P21 (A, C) protein expression levels in the control group (NC) and experimental group of the aging model of primary antler cartilage cells after H2O2 treatment for 2 hours (P<0.001).

[0022] Figure 6 Cell proliferation results of the uridine pretreatment group (the group in which uridine pretreatment was followed by cell senescence) and the uridine recovery group (the group in which uridine treatment had already caused senescence). NC: control group; H2O2: senescence group; 1mM+H2O2, 2mM+H2O2: uridine pretreatment group;

[0023] H2O2+1mM, H2O2+2mM: uridine recovery group.

[0024] Figure 7 Cell SA-β-GAL staining results (A) and positive staining rate (B) of uridine pretreatment group and uridine recovery group. Figure A (a): control group; Figure A (b): aging group; Figure A (c): uridine pretreatment group; Figure A (d): uridine recovery group. In Figure B, NC: control group;

[0025] H2O2: aging group; 2mM+H2O2: uridine pretreatment group; H2O2+2mM: uridine recovery group.

[0026] Figure 8 Comparison of the expression abundance of cell marker genes between the uridine pretreatment group and the uridine recovery group. NC: control group; H2O2: aging group; 2mM+H2O2: uridine pretreatment group; H2O2+2mM: uridine recovery group. DETAILED DESCRIPTION

[0027] To better understand the present invention, the invention is described in more detail and in detail below through specific embodiments in conjunction with the relevant drawings. The following embodiments facilitate the present invention to be implemented in many different forms and are not limited to the embodiments described herein. The purpose of providing these embodiments is to make the disclosure of the present invention more thorough and comprehensive. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by technicians in the technical field of the present invention. The experimental methods in the following embodiments, unless otherwise specified, are conventional methods. The experimental materials used in the following embodiments, unless otherwise specified, are purchased from conventional biochemical reagent companies. The quantitative experiments in the following embodiments were all repeated three times, and the results were averaged. In the statistical analysis of the experimental results in the following embodiments, P < 0.05 indicates that the difference is statistically significant, P < 0.01 indicates that the difference is significant, and P < 0.001 indicates that the difference is extremely significant.

[0028] Example 1: Effect of H2O2 on the activity of primary antler cartilage cells and determination of the half effective concentration.

[0029] (1) Determination of the half-effective concentration of H2O2-induced cell senescence using CCK8 method

[0030] Experimental methods:

[0031] The primary cells that were subcultured to the third generation after recovery were inoculated in a 96-well plate at a density of 2000 / well, with 100μl complete medium in each well. The 96-well plate was taken out after culturing (placed in an incubator at 37°C, 5% CO2) for 24 hours. The experiment was divided into a control group and an experimental group. The control group was added with serum-free medium, and the experimental group was added with serum-free medium containing 100μM, 200μM, 400μM, 800μM, and 1600μM H2O2 concentrations, respectively. After induction for 0.5h, 1h, 1.5h, and 2h, all were replaced with complete medium. After continuing to culture for 72h, the supernatant was discarded, and 110μl CCK-8-serum-free medium solution (serum-free medium: CCK8=10:1) was added, and incubated in the incubator for 1-2h. The absorbance value at a wavelength of 450nm was read using an enzyme marker.

[0032] Experimental results:

[0033] The effects of different concentrations of H2O2 on the survival rate of primary antler cartilage cells were significantly different, and were inversely correlated with the concentration ( Figure 1 ). Compared with the control group, the mean cell survival rate decreased to 79% when the concentration of H2O2 was 200μM; when the concentration was 400μM, the cell survival rate was about 50%, and the cell proliferation was significantly slowed down; when the concentration of H2O2 reached 800μM and above, the cells stopped growing, most of the cells died, and the cell bodies of the remaining cells shrank ( Figure 2 D). The experimental results were fitted by nonlinear regression, and the formula was y = 1.053-0.001501*x+5.357e-0.07*x 2 , R 2 =0.9863. The above results show that the H2O2 concentration of 400 μmol / L is the half-effective concentration of cell senescence, and the treatment time is selected as 2 hours.

[0034] (2) Determination of the half-effective concentration of H2O2-induced cell senescence by β-galactosidase (SA-β-GAL) staining analysis

[0035] Experimental methods:

[0036] The same batch of primary cells as in Example 1(1) of the experimental method of this study were cultured at 2×10 5The cells were inoculated at a density of 100 cells / well in a 6-well plate and divided into a control group and an experimental group. The H2O2 treatment method for the control group and the experimental group is shown in the experimental method section of Example 1 (1). After 72 hours of cell culture, the cells were stained using a SA-β-GAL staining kit. Senescent cells appear blue under a microscope. Three groups of fields (10×20) were randomly selected for counting and the positive staining rate was calculated (positive staining rate = number of positive cells / total number of cells).

[0037] Experimental results:

[0038] SA-β-gal staining can be used to evaluate the aging state of cells by showing the activity of intracellular β-galactosidase. Figure 2 ) showed that compared with the control group ( Figure 2 A), H2O2 concentration of 200 μM can upregulate the β-galactosidase activity of primary cells, and some cell nuclei begin to show a lighter sky blue color ( Figure 2 B right); When the concentration reaches 400 μM, the sky blue color around the nucleus of senescent cells is obvious, the border is blurred, and the normal spindle shape changes into irregular oval, slender and other senescent characteristic shapes ( Figure 2 C right); at concentrations of 800 μM and above, most cells have shrunk and died ( Figure 2 D right). Calculation of cell positive staining rate showed that 200μM or 400μM H2O2 concentration could make the positive staining rate of senescent cells reach the commonly used ideal positive staining rate standard (70%, Figure 3 ). Combined with the experimental results (1), the subsequent experiments selected 400 μM H2O2 to treat cells for 2 hours to construct a primary antler cartilage cell aging model.

[0039] Example 2: Effect of H2O2 on the expression of key marker genes in the TP53-CDKN1A (P21) axis controlling aging in primary antler cartilage cells

[0040] (1) Detection of transcriptional expression changes of aging-related marker genes TP53 and P21 using RT-qPCR technology

[0041] Experimental methods:

[0042] For the method of constructing the experimental group for aging induction of primary antler cartilage cells, refer to the experimental method of Example 1 (1). A control group was set up in the experiment. The primary cells were seeded in a 6-well plate and grown to 70% to 80% confluence. After being treated with 400 μM H2O2 for 2 hours, the complete culture medium was replaced and cultured for 72 hours. Total RNA was extracted using an RNA extraction kit. The concentration of the extracted total RNA was measured using NanoDrop One to determine whether the prepared RNA had the integrity required by the experiment (A260 / A280=2.1, A260 / A230=2.0). A conventional cDNA synthesis kit was then used to synthesize cDNA after removing the genomic DNA. Store in a -20°C refrigerator for later use.

[0043] Based on the TP53 gene sequence of red deer (accession number: XM_043903734.1) and the P21 gene sequence (accession number: XM_043907314.1) in GenBank, qPCR primers were designed online using NCBI-Primers-BLAST and submitted to Harbin Ruiboxingke (Northeast) Biotechnology Co., Ltd. for synthesis (primer details are shown in Table 1).

[0044] Table 1 Primer information used in RT-qPCR detection

[0045]

[0046] In this experiment, GADPH was used as the internal reference gene, 400 μM H2O2 was selected to induce senescence in primary cells, and the cDNA of cells in the control group and the experimental group was used as template to perform qPCR on the transcriptional expression abundance of TP53 and P21 genes (Roche 96) detection. The reaction system (20 μL) of each sample was as follows: 10 μL of 2×SYBR Green QPCR Master Mix, 0.5 μL of upstream and downstream primers, 1 μL of template, and 8 μL of ddH2O. PCR amplification program: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 10 s, 60℃ annealing for 30 s, for a total of 45 cycles; each sample had 3 independent replicates. 2 -ΔΔCt RT-qPCR data were analyzed and the relative expression levels of marker genes between the control group and the experimental group were calculated.

[0047] Experimental results:

[0048] RT-qPCR detected that the transcriptional expression of related marker genes TP53 and P21 mRNA in H2O2-induced senescent cells was upregulated ( Figure 4). The TP53-P21 axis is the core pathway for controlling cell aging in response to oxidative stress. The results of RT-qPCR detection of the transcriptional expression of the core markers TP53 and P21 in this pathway showed that after 2 hours of exposure to H2O2 concentration of 400μM, the transcriptional levels of TP53 and its downstream target gene P21 in the experimental group were significantly upregulated compared with the control group (p<0.001), and the expression level of P21 was about 3 times that of the control group ( Figure 4 B), cells are in a state of senescence.

[0049] (2) Detection of protein expression changes of senescent cell marker genes TP53 and P21 using Western Blotting

[0050] Experimental method: 400μM H2O2 was selected to induce senescence in primary cells, and a control group was set up in the experiment. Cells were cultured and collected, cell lysis solution was added, and protein samples were prepared by low-temperature centrifugation. Using the NanoDrop One instrument, the BSA280 method was selected to detect the protein concentration. The protein sample was boiled at 100℃ for 10min, and the supernatant was taken for protein gel electrophoresis (180V, 25min) after high-speed centrifugation. After electrophoresis, the membrane was transferred (40V, 90min). It was blocked with 5% skim milk, and after sequential incubation with antibodies, The CLX dual-color infrared laser imaging system scans the membrane, collects and analyzes data.

[0051] The optical density of Western Blot electrophoresis was analyzed by Image J software. GraphPad Prism9.0 software was used to analyze and plot the data, and one-way analysis of variance and t-test were used for comparison. P < 0.05 indicated that the difference was statistically significant, P < 0.01 indicated that the difference was significant, and P < 0.001 indicated that the difference was extremely significant.

[0052] Experimental results:

[0053] The results of the detection of the protein levels of the key marker genes of the TP53-P21 axis showed that after treatment with 400 μM H2O2, the protein expression levels of the aging marker genes TP53 and P21 in the primary cells were significantly upregulated in the experimental group compared with the control group ( Figure 5 ). Combined with the RT-qPCR results, it was suggested that H2O2 induced cells to upregulate the transcription and protein expression levels of the TP53 gene and its downstream regulated P21 gene.

[0054] Example 3: Application of a novel cartilage tissue cell aging model in delaying cell aging with uridine

[0055] (1) Effects of uridine on cell activity in aging models

[0056] This experiment examined the effect of uridine on cell activity in a novel cartilage cell aging model by measuring the effect of uridine on the proliferation of induced senescent cells and pre-treated and then induced senescent cells.

[0057] Experimental methods:

[0058] In this experiment, the concentration of H2O2, the inducer of cell senescence, was 400 μM, and the induction time was 2 h; the concentration of uridine was 1 mM and 2 mM (the appropriate concentration screened by the previous experiment), and the cell treatment time was 48 h. Deer antler chondrocytes were seeded in 3 plates of 96-well plates at a density of 3000 cells / well, and divided into blank group, control group, senescence group, uridine pretreatment group (cell senescence group induced after uridine pretreatment) and uridine recovery group (senescent cell group treated with uridine), with 4 replicates in each group. After 24 h of culture, the medium was aspirated, and complete medium was added to the control group, senescence group, and uridine recovery group. After the uridine pretreatment group was pretreated with complete medium containing uridine, the medium was aspirated again. Except for the control group, each experimental group was induced with medium containing 400 μM H2O2 for 2 h, and complete medium was added to the senescence group and uridine pretreatment group, and complete medium containing uridine was added to the uridine recovery group. After 48 h, CCK-8 was added for cell proliferation detection.

[0059] Experimental results:

[0060] Effects of uridine treatment on proliferation of aged antler chondrocytes Figure 6 As shown in the results, whether uridine was used to pre-treat antler chondrocytes and then induce senescence, or uridine was used to restore senescent antler chondrocytes, the cell proliferation of the uridine-added group was significantly improved compared with the senescent group, and the effect of 2mM uridine concentration was better. This result preliminarily proves that uridine treatment has a delaying effect on the senescence process of antler cells. Figure 6 As a result, 2 mM uridine concentration was selected for subsequent senescent cell recovery experiments.

[0061] (2) SA-β-GAL staining results of uridine-treated aged antler chondrocytes and pre-treated antler chondrocytes induced to age

[0062] Experimental methods:

[0063] The cells were stained using the β-galactosidase staining kit according to the experimental method of Reference Example 1(2).

[0064] Experimental results:

[0065] The results of SA-β-GAL staining of cells showed that compared with the aging group, the positive staining rates of the uridine pretreatment group and the uridine recovery group decreased significantly ( Figure 7 A), data analysis showed significant differences compared with the aging group ( Figure 7B) It is worth noting that although the positive infection rate of both is still above 50%, Figure 7 A(c, d) and Figure 7 Compared with A(b), it can be clearly seen that the staining intensity of SA-β-GAL positively stained cells in the uridine recovery group and the uridine pretreatment group was much lower than that in the aging group.

[0066] (3) Changes in mRNA expression of senescence marker genes TP53 and P21 in uridine-treated senescent antler chondrocytes and senescent antler chondrocytes induced by pretreatment

[0067] Experimental methods:

[0068] Deer antler chondrocytes were cultured at 5×10 5 The cells were inoculated in 6-well plates and cultured for 24 h. The cell treatment method was the same as that of Example 3 (1). The RNA extraction, cDNA synthesis and qPCR detection of the transcriptional expression abundance of TP53 and P21 genes were the same as those of Example 2 (1). GADPH was used as the internal reference gene and the same qPCR reaction system was used. The primers in Table 1 were used to perform qPCR amplification of the TP53, P21 and GADPH genes to detect changes in their mRNA expression.

[0069] Experimental results:

[0070] RT-qPCR results showed that in the uridine recovery group, the transcription levels of TP53 and P21, core markers of senescent cells, were significantly downregulated compared with those in the senescent group, and TP53 even reached the same level as that in the control group ( Figure 8 A, B), which shows that after uridine treatment of aged antler chondrocytes, the cell growth activity is improved, regardless of whether it is the uridine pretreatment group ( Figure 8 AB 1mM+H2O2, 2mM+H2O2) or uridine recovery group ( Figure 8 AB H2O2+1mM, H2O2+2mM) both have the effect of delaying cell aging.

[0071] In summary, the novel cell aging model provided by the present invention can be used to accurately screen and evaluate the anti-aging effects of small molecule compounds on primary red deer antler cartilage cells.

[0072] The content of the present invention is not limited to the content of the above-mentioned embodiments. The combination of one or several specific embodiments can also achieve the purpose of the invention.

[0073] The above-mentioned embodiments only express several implementation methods of the present application. Although the description is relatively specific and detailed, it cannot be understood as limiting the scope of the invention patent. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present application, which all belong to the protection scope of the present application. Therefore, the protection scope of the patent of this application shall be based on the attached claims.

Claims

1. A method for establishing an aging model of primary cells of red deer antler cartilage, characterized in that: include: 100-1600 μM H2O2 was added to the complete culture medium to induce the primary cells of the antler cartilage tissue of red deer for 0.5-2 h. The culture medium was replaced with complete culture medium and cultured for 72 h. The cell senescence was analyzed by CCK-8 method combined with β-galactosidase (SA-β-GAL) staining. The senescence half-effective concentration of H2O2 was determined to be 400 μM, and the induction time was 2 h. Significant changes in the transcription and protein expression levels of cell senescence marker genes were obtained.

2. The method for establishing the primary cell aging model according to claim 1, characterized in that: The primary cells are derived from red deer, and the cells are from antler cartilage tissue in the rapid proliferation period. The complete culture medium components include: DMEM, 10% fetal bovine serum, 1% penicillin / streptomycin, and the serum-free culture medium components include: DMEM, 1% penicillin / streptomycin.

3. The method for establishing the primary cell aging model according to claim 1, characterized in that: The cells were induced with 400 μM H2O2 for 2 h, replaced with complete culture medium, and cultured for another 72 h. CCK-8 screening was used, and SA-β-GAL staining was used to analyze cell senescence. It was determined that the senescence half-effective concentration of primary cells of red deer antler cartilage was 400 μM, and the induction time was 2 h.

4. The method for establishing the primary cell aging model according to claim 1, characterized in that: The cells were induced with 400 μM H2O2 for 2 h, replaced with complete culture medium, and cultured for another 72 h. Real-time quantitative PCR (RT-qPCR) and Western Blotting methods were used to detect changes in transcription and protein expression of key marker genes of the TP53-CDKN1A (P21) axis that controls aging in cells to confirm that the modeling was successful.

5. A primary cell aging model of red deer antler cartilage, characterized in that: The method is constructed by the method described in any one of claims 1 to 4.

6. Use of the red deer antler cartilage primary cell aging model described in claim 5 in screening small molecule compounds that delay cell aging.

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