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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 Caspase-7 hammerhead ribozyme on target genes in primary hepatocytes. Methods The eukaryotic expression plasmid pRz333 containing Cas-pase-7 hammerhead ribozyme was constructed. After transfection into primary hepatocytes, the expression of Caspase-7 mRNA was up-regulated by TNF-α and actinomycin-D. RT-PCR and Western blotting were used to detect the expression of Caspase-7mRNA and proteasome. The apoptosis rate of each group of cells was detected by flow cytometry. Results Compared with the induction group, the expression of Caspase-7 mRNA decreased by 29.34%, the activity of Caspase-7 decreased by 32.12% and the apoptosis rate decreased by 11.75%. Conclusion Anti-Caspase-7 hammerhead ribozyme can specifically cut target genes in primary hepatocytes, down-regulate mRNA and protein expression of target genes and reduce apoptosis of hepatocytes.