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目的观察小鼠半胱天冬氨酸蛋白酶-12(caspase-12)基因特异性小干扰RNA (siRNA)构建的表达载体pRNAT-casp12对caspase-12基因的抑制及其对内质网应激介导的小鼠肝细胞凋亡的影响。方法以小鼠肝细胞株Hepa 1-6为靶细胞,利用脂质体与重组质粒pRNAT- caspl 2共转染,分别在转染24、48和72 h后收集细胞,采用实时荧光定量PCR分析和Western印迹检测caspase-12的表达;利用毒胡萝卜素(TG)诱导细胞,建立内质网应激介导的细胞凋亡模型,通过DNA梯带凝胶电泳检测,选出适合的诱导时间;TG诱导已转染了pRNAT-casp12的干扰组细胞后,以空质粒转染组为对照,利用Western印迹检测caspase-12蛋白表达水平的变化,通过DNA梯带凝胶电泳、流式细胞仪、Hoechst 33258染色等方法检测细胞凋亡,观察caspase-12 siRNA对细胞凋亡的影响。结果pRNAT-casp12转染细胞24、48和72 h后,caspase-12 mRNA的水平分别下降了45.6%、72.5%和59.5%;caspase-12蛋白表达下降了17.1%、37.3%和60.1%;2μmol/L TG处理细胞30 h后,成功诱导细胞凋亡;与对照组相比,干扰组细胞经TG诱导后,caspase-12蛋白表达水平下降了54.6%,流式细胞仪检测发现早期调亡率下调了51.4%(P<0.01)。结论小鼠caspase-12 siRNA对Hepa 1-6细胞caspase-12基因的表达具有显著的抑制作用,能够明显阻抑内质网应激介导的凋亡,有望发展成为新一代抗凋亡药物。
Objective To observe the inhibitory effect of mouse caspase-12 gene-specific small interfering RNA (siRNA) on the expression of caspase-12 gene and its effect on the endoplasmic reticulum Effect of Apoptosis in Hepatic Cells Induced by. Methods Mouse Hepatoma cell line Hepa 1-6 was used as target cells and co-transfected with pRNAT-caspl 2 by liposome. The cells were harvested at 24, 48 and 72 h after transfection respectively. Real-time fluorescent quantitative PCR And Western blotting were used to detect the expression of caspase-12. Endodermal stress-induced apoptosis model was established by using thapsigargin (TG) -induced cells. The suitable induction time was selected by DNA ladder gel electrophoresis. TG induced the transfected pRNAT-casp12 interference group of cells, empty plasmid transfected group as a control, Western blotting detection of caspase-12 protein expression changes by DNA ladder gel electrophoresis, flow cytometry, Hoechst 33258 staining and other methods to detect apoptosis, observed caspase-12 siRNA on apoptosis. Results The caspase-12 mRNA levels decreased by 45.6%, 72.5% and 59.5%, and decreased by 17.1%, 37.3% and 60.1% in pRNAT-casp12 transfected cells at 24, 48 and 72 h, respectively. / L TG treatment of cells after 30 h, successfully induced apoptosis; compared with the control group, the expression of caspase-12 protein decreased 54.6% after induction by TG, the early apoptosis rate was detected by flow cytometry Down 51.4% (P <0.01). Conclusions The mouse caspase-12 siRNA significantly inhibits the expression of caspase-12 gene in Hepa 1-6 cells and significantly inhibits endoplasmic reticulum stress-mediated apoptosis. It is expected to develop into a new generation of anti-apoptotic drugs.