| PartⅠ: Induction and phenotype comparison of different proportions of senescent cells in rat pancreatic AR42J cellsObjective: To induce different proportions of senescent cells in rat pancreatic exocrine cell line AR42 J cells by different concentrations of D-galactose for 72 hours,and compare the basic phenotypes of the cells in each group.Methods: Rat pancreatic AR42 J cells were administered with D-galactose of different concentrations for 72 hours.After the intervention was completed,the cell morphology of each group was observed under a light microscope.After attachment,senescence-associated β-galactosidase staining(SA-βG)was performed to compare the proportion of positive cells in each group,that is,the proportion of senescent cells;according to cell morphology and SA-βG staining,the proportions of senescent cells with significant differences were screened.D-galactose was used to induce the selection of 4 groups of senescent cells of different proportions.After inoculating cells onto the slides,the expression levels of p19 ARF and Ki67 in each group were detected by cellular immunofluorescence experiments;the cell cycle of cells in each group was detected by flow cytometry;and the level of senescence-related secretion phenotype(SASP)of each group was detected by immunoblotting experiments.Results: According to the changes of cell morphology and SA-βG staining,the Dgalactose concentrations screened were 0,10,20,and 40 mg / ml,and named as D0,D10,D20,and D40,respectively.The proportion of induced senescent cells was 6.22%,12.63%,24.63%,47.34%,there is no significant difference between D0 and D10 groups,which is not statistically significant(P> 0.05),and the remaining differences are statistically significant(P <0.05);as the proportion of senescent cells increases,the expression level of p19 arf increased gradually,and the expression level of ki67 gradually decreased,the difference was statistically significant(P <0.05);the G1 phase of the cell cycle was blocked as the proportion of senescent cells increases,and the proportion of cells in the G2/M phase decreased as the proportion of senescent cells increases,and the difference increased statistically(P <0.05));From D0 to D40 group,the expression level of SASP gradually increased,and the difference was statistically significant(P <0.05).Conclusion: D-galactose can induce the generation of senescent cells of rat exocrine lineage cells AR42 J with different proportions,and the higher the proportion of senescent cells,the higher the level of SASP expression.PartⅡ: Establishment and comparison of cell injury of rat pancreatic AR42 J cells with different proportions of senescent cellsObjective: After D-galactose induced different proportions of senescent cells in AR42 J,to induce cell injury with L-arginine for 24 hours,and to compare the injury of senescent cells with different proportions.Methods: The rat pancreatic exocrine cell line AR42 J was inoculated into a 96-well plate,and different concentrations of L-arginine were applied for 24 hours,cytotoxicity test CCK-8 kit was used to detect the cell activity after each concentration of L-arginine,and select a suitable L-arginine concentration.AR42 J cells were divided into 4 groups: LD0 group(L-Arg/D-gal 0mg/ml),LD10 group(L-Arg/D-gal 10mg/ml),LD20 group(L-Arg/D-gal 20mg/ml),LD40 group(L-Arg/D-gal 40mg/ml);LD0 group:induced with 0 mg/ml D-galactose for 72 hours,and then applied L-arginine for 24 hours to induce cell injury;LD10 group: induced with 10 mg/ml D-galactose for 72 hours,and then applied L-arginine for 24 hours to induce cell injury;LD20 group:induced with 20 mg/ml D-galactose for 72 hours,and then applied L-arginine for 24 hours to induce cell injury;LD40 group: induced with 40 mg/ml D-galactose for 72 hours,and then applied L-arginine for 24 hours to induce cell injury;after centrifugation of the supernatant,the activity level of α-amylase(α-AMY)in the supernatant of each group was detected;the apoptosis and necrosis of the cells were detected by flow cytometry;the mitochondrial membrane potential kit was used to detect the changes of mitochondrial membrane potential of cells in each group;correlation analysis was used to compare the correlation between SASP and the injury status of each group after induced injury.Results: The concentration of L-arginine selected according to the results of cytotoxicity experiments was 10 mg/ml.After inducing injury to different proportions of senescent cells,as the proportion of senescent cells increased,the activity of α-AMY in the supernatant gradually increased.The levels of apoptosis and necrosis gradually increased,and the proportion of mitochondrial membrane potential loss gradually increased,the differences were statistically significant(P <0.05);SASP was positively correlated with cell injury,and the differences were statistically significant(P <0.05).Conclusion: After the induction of cell injury,with the increase of the proportion of senescent cells,the cell injury is heavier,which is positively related to the level of SASP.After cell senescence,SASP will aggravate the degree of cell injury when it is injuryd by external stimuli.Part Ⅲ: The role of p53 pathway regulating senescencerelated secretory phenotype in the injury of rat pancreatic cell line AR42 J cellsObjective: To induce the senescence of rat pancreatic cell line AR42 J cells,use p53 inhibitors to intervene the level of senescence-related secretory phenotype(SASP),and then use l-arginine to induce cell injury,to investigate the role of p53 in SASP in pancreatic cell injury and its possible mechanism.Methods: AR42 J cells of rat pancreatic exocrine cell line were treated with 40mg/ml d-galactose for 72 hours to induce senescence,and then inoculated into 96-well plates,they were given different concentrations of p53 inhibitor PFT-α for 2 hours and24 hours,cytotoxicity test CCK-8 was used to detect the cell viability of each group,and the optimal concentration of PFT-α and the intervention time were screened.AR42 J cells were divided into three groups: group D(D-gal group),group LD(L-arg/D-gal group),group LDT(L-arg/D-gal/Treatment group);group LDT: after inducing cell senescence with 40 mg/ml d-galactose for 72 hours,a p53 inhibitor PFT-α was given at a certain time and dose,after the intervention,10 mg/ml l-arginine was used for 24 hours to induce injury;group LD: after inducing cell senescence with 40 mg/ml dgalactose for 72 hours,a solvent intervention was given for the same time and then 10mg/ml L-arginine was used for 24 hours to induce injury;group D: after 72 hours of induction of cell senescence with 40 mg/ml d-galactose,a solvent control was given;after centrifuging the supernatant of each group,the activity level of α-amylase(α-AMY)in the supernatant was detected;the apoptosis and necrosis of cells were detected by flow cytometry;the mitochondrial membrane potential kit was used to detect the changes of mitochondrial membrane potential in each group of cells.Besides,AR42 J cells were further divided into three groups: group N,group D,group P,group N:without any intervention,only the solvent was given for control;group D: after 40-mg/ml d-galactose was used to induce cell senescence,given solvent intervention with p53 inhibitor PFT-α;Group P: PFT-α was administered after 40-mg/ml D-galactoseinduced cell senescence;the expression levels of senescence-related secretory phenotype(SASP)and the expression levels of p53 pathway proteins p53,m TOR,p-4EBP1 and IL-1α were detected by western blot.Results: According to the results of cytotoxicity experiments,the intervention concentration of PFT-α was 0.4 μM,and the intervention time was 24 hours.After PFT-α intervention,the cell injury was relieved,the activity of α-AMY in the supernatant was reduced,the apoptosis and necrosis of the cells were relieved,and the loss of mitochondrial membrane potential was improved,the differences were statistically significant(P <0.05).After the intervention of PFT-α,the SASP level of senescent cells was significantly reduced,and the difference was statistically significant(P <0.05);the expression levels of p53,m TOR,p-4EBP1 and IL-1α were significantly reduced,and the differences were statistically significant(P <0.05).Conclusion: L-arginine-induced injury of senescent cells of AR42 J can be significantly improved by reducing SASP levels in senescent cells.Application of p53 inhibitors can reduce SASP levels in senescent cells,thereby reducing injury to senescent cells. |
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