中文摘要：

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Hepa1-6为靶细胞，利用脂质体与重组质粒pRNAT-casp-2共转染，分别在转染24、48和72h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b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、Hoechst33258染色等方法检测细胞凋亡，观察caspase-12siRNA对细胞凋亡的影响。结果pRNAT—casp12转染细胞24、48和72h后，caspase-12mRNA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30h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-12siRNA对Hepa1—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。

英文摘要：

Objective To investigate the suppressive effect of small interfering double-stranded RNA（siRNA） on caspase-12 expression and its influence on endoplasmic reticulum（ER） stress-induced apoptosis in mouse hepatoma cell line. Methods The plasmid pRNAT-casp12 was transfected into mouse hepatoma cell line, Hepa 1-6, for 24, 48 and 72 hours respectively. The transcription and expression levels of caspase -12 were analyzed by real-time polymerase chain reaction and Western bolot respectively. Cells transfected with blank pRNAT-H1. 1 Neo vector were used as controls. Hepa 1-6 was treated with thapsigargin（TG） to induce ER stress-mediated apoptosis. The incubation time of TG for inducing apoptosis was determined by agarose gel electrophoresis showing DNA fragmentation. Then Hepa 1-6 cells were transfected with pRNAT-casp12 and then treated with 2 μmol/L TG for 30 hours. The changes of the protein levels of caspase-12 were analyzed by Western blot. The percentage of apoptotic cells labelled with annexin V-fluoresce in isothiocyanate （FITC） was measured by flow cytometry . The inhibition of apoptosis was evaluated by agarose gel electrophoresis and staining witht Hoechst 33258. Results After transfection with pRNAT-casp12 for 24, 48 and 72 hours, the tran- n level of caspase 12 gene in the cells was knockdowned by 45.6%, 72.5% and 59.5% respec- while the amount of procaspase-12 protein in Hepa 1-6 cells decreased 17. 1%, 37. 3% and respectively compared with the mocktransfection. After treatment with TG, the expression of procaspase-12 was reduced by 54.6~ in the cells transfected with pRNAT-casp12. Moreover, treat- ment with pRNAT-casp12 suppressed TG-induced morphologic changes of ER stress apoptosis and reduced the percentage of apoptotic cells by 51.4% compared with the mock transfection （P〈0.01）. Conclusions These results suggest that caspase-12 siRNA effectively down-regulate the expression of caspase-12 in Hepa 1 6 cells and inhibit ER stress-induced cell apoptosis. Furthermore, our results su