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目的探讨亚硫酸钠对人正常二倍体肝细胞(HL-7702)的损伤作用及程序性坏死相关蛋白受体相互作用蛋白1(RIP1)表达的影响。方法将处于对数生长期的HL-7702细胞分别暴露于含终浓度为0(对照)、0.01、0.1、1、10 mmol/L亚硫酸钠的培养基中培养2、4、12、24、48 h。采用CCK-8法检测细胞活性,采用全自动生化分析仪检测细胞上清液中丙氨酸氨基转移酶(ALT)、天冬氨酸氨基转移酶(AST)以及乳酸脱氢酶(LDH)的活力,采用Western Blot法检测细胞RIP1蛋白的表达水平。结果与对照组相比,1、10 mmol/L亚硫酸钠染毒不同时间的HL-7702细胞存活率降低,差异有统计学意义(P<0.05);且随着亚硫酸钠染毒浓度的升高,HL-7702细胞的存活率呈下降趋势。与对照组相比,仅10 mmol/L亚硫酸钠染毒24、48 h HL-7702细胞上清液中的ALT活力较高;1 mmol/L亚硫酸钠染毒4、24 h及10 mmol/L亚硫酸钠染毒2、4 h HL-7702细胞上清液中的AST活力较低,而10 mmol/L亚硫酸钠染毒24、48 h HL-7702细胞上清液中的AST活力较高;10 mmol/L亚硫酸钠染毒2、4 h HL-7702细胞上清液中的LDH活力较低,而染毒24、48 h HL-7702细胞上清液中的LDH活力较高,差异均有统计学意义(P<0.05)。与对照组相比,各浓度亚硫酸钠染毒组HL-7702细胞中RIP1蛋白的表达水平均较高,差异多有统计学意义(P<0.05)。结论程序性坏死可能参与了亚硫酸钠引起的肝细胞死亡,但其可能机制还有待于进一步研究。
Objective To investigate the effect of sodium sulfite on the injury of human normal diploid hepatocytes (HL-7702) and the expression of the protein of programmed death-related receptor 1 (RIP1). Methods HL-7702 cells in logarithmic growth phase were exposed to culture media containing 0.01, 0.1, 1, 10 mmol / L sodium sulfite at the final concentration of 0 (control), 2,4,12,24,48 h . The cell viability was determined by CCK-8 assay. The contents of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) in the cell supernatant were detected by automatic biochemical analyzer Viability. Western Blot method was used to detect the expression level of RIP1 protein. Results Compared with the control group, the survival rate of HL-7702 cells treated with 1, 10 mmol / L sodium sulfite decreased at different time points (P <0.05). With the increase of sodium sulfite concentration, HL -7702 cells showed a decreasing trend of survival. Compared with the control group, ALT activity in supernatant of HL-7702 cells treated with 10 mmol / L sodium sulfite for 24 and 48 h was higher than that of the control group. Sodium sulfite (1 mmol / L) was used for 4, 24 and 10 mmol / L sodium sulfite staining The activities of AST in supernatant of HL-7702 cells treated with 2,4-dichlorobenzene were lower than those of cells treated with 10 mmol / L sodium sulfite for 24,48 h, while the activities of 10 mmol / L sodium sulfite The LDH activity of HL-7702 cells supernatant was lower at 2,4 h after exposure, while the LDH activity of HL-7702 cells supernatant at 24 h and 48 h after exposure was significantly higher (P < 0.05). Compared with the control group, the expression levels of RIP1 protein in HL-7702 cells treated with sodium sulfite were significantly higher than those in the control group (P <0.05). Conclusion Programmed necrosis may be involved in the death of hepatocytes induced by sodium sulfite, but its possible mechanism remains to be further studied.