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探讨内源性胱硫脒-γ-裂解酶/硫化氢(CSE/H2S)对人肝癌HepG2细胞凋亡的调控作用。采用MTT法和台盼蓝染色法检测细胞增殖;亚甲基蓝法检测细胞内源性H2S的产生;PI/Hoechst双染法检测细胞凋亡;荧光探针法检测细胞内超氧阴离子及ROS水平;OxiSelect Total Glutathione Assay试剂盒检测细胞内还原型谷胱甘肽(GSH)水平;Western blotting检测activated-caspase 3、p-AKT和Nrf2表达。结果显示,CSE抑制剂DL-炔丙基甘氨酸(PAG)和CSE siRNA可明显降低细胞内H2S水平,可呈剂量和时间依赖性地抑制HepG2细胞增殖。PAG和CSE siRNA均可明显增加HepG2细胞凋亡率以及细胞内超氧阴离子和ROS的水平,降低细胞内GSH水平。CSE siRNA引起的ROS水平升高及GSH水平降低可被重组质粒pcDNA 3.1/myc-His()-CSE恢复。CSE siRNA可促进caspase 3的活化,但不会影响p-AKT及Nrf2蛋白的表达。结果表明,内源性CSE/H2S系统可通过氧化应激调控HepG2细胞的增殖及凋亡。
To investigate the regulatory effect of endogenous cystathionine-γ-lyase / hydrogen sulfide (CSE / H2S) on human hepatoma HepG2 cells. MTT and trypan blue staining were used to detect cell proliferation; methylene blue assay was used to detect the production of endogenous H2S; PI / Hoechst double staining was used to detect apoptosis; fluorescence probe was used to detect intracellular superoxide anion and ROS levels; OxiSelect Total Glutathione Assay kit was used to detect intracellular reduced glutathione (GSH) levels. The expressions of activated-caspase 3, p-AKT and Nrf2 were detected by Western blotting. The results showed that CSE inhibitor DL-propargylglycine (PAG) and CSE siRNA could significantly reduce intracellular H2S levels and inhibit HepG2 cell proliferation in a dose-and time-dependent manner. Both PAG and CSE siRNA could significantly increase the apoptosis rate of HepG2 cells and the level of intracellular superoxide anion and ROS, as well as decrease the intracellular GSH level. CSE siRNA induced ROS levels increased and GSH levels decreased by recombinant plasmid pcDNA 3.1 / myc-His () - CSE recovery. CSE siRNA can promote caspase 3 activation, but does not affect the expression of p-AKT and Nrf2 protein. The results show that endogenous CSE / H2S system can regulate the proliferation and apoptosis of HepG2 cells through oxidative stress.