抗Caspase-7锤头状核酶在原代肝细胞内的切割活性研究  

Research of cleavage activity of hammerhead ribozyme targeting caspase-7 in primary cultured liver cells

作　　者：张韦华 谢青[2] 姜山[2] 俞红[2] 臧国庆[1] 

机构地区：[1]上海交通大学附属第六人民医院传染科,上海200233 [2]上海交通大学附属瑞金医院传染科,上海200025

出　　处：《中国现代医学杂志》2009年第20期3063-3066,共4页China Journal of Modern Medicine

基　　金：上海市卫生局青年科研基金项目(No:054Y03)

摘　　要：目的研究抗Caspase-7锤头状核酶在原代肝细胞内对靶基因的切割活性。方法构建含抗Cas-pase-7锤头状核酶的真核表达质粒pRz333。将其转染原代肝细胞后,用TNF-α和放线菌素D上调细胞内Caspase-7 mRNA的表达。RT-PCR和免疫印迹法检测Caspase-7mRNA和蛋白酶原的表达。用流式细胞仪检测各组细胞的凋亡率。结果与诱导组相比,核酶组肝细胞内Caspase-7 mRNA的表达降低了29.34%;Caspase-7蛋白酶原含量下降了32.12%;细胞凋亡率减低了11.75%。结论抗Caspase-7锤头状核酶在原代肝细胞内能够特异性切割靶基因,使目的基因的mRNA和蛋白质表达下调,并能够减少肝细胞凋亡。[ Objective ] To study the cleavage activity of hammerhead ribozyme against caspase-7 in primary liver cells. [Methods] Eukaryotie expression plasmid pRz333 was constructed by molecular cloning technics. The pRz333 and empty pEGFP were transfected into cultured liver cells respectively. Then TNF-α and Act-D were used to induce cell apoptosis so as to enhance the level of caspase-7 mRNA. The levels of caspase-7 mRNA was detected by RT-PCR, and the amount of pro-caspase-7 protein was examined by western blot. Cell apoptosis was examined by flow cytometry. [ Results ] Compared with revulsive-group, the level of caspase-7 mRNA of ribozyme-group was markedly decreased by 29.34%, the amount of pro-caspase7 was also decreased by 32.12%, and the ratio of cell apoptosis in experiment group was also decreased by 11.75%. [Conclusions] Rz333 can site-specifically cleave caspase-7 mRNA in primary cultured liver ceils, and down-regulate the expression of pro-caspase-7 protein. By inhibiting activity of caspase-7, Rz333 can reduce cell apoptosis.

关 键 词：锤头状核酶 CASPASE-7 催化切割 凋亡 

分 类 号：R-331[医药卫生] R318.14