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

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.