摘要
目的 :观察人重构型caspase 6基因在Hep 2细胞中的表达及其对转染的Hep 2细胞的作用 .方法 :用RT PCR方法获取人caspase 6全长cDNA ,经重组PCR构建大小亚基次序颠倒的重构型caspase 6 (re caspase 6 ,简称rcasp6 ) .将所获重组基因克隆入真核表达载体pCMV ,转染Hep 2细胞 .用间接免疫荧光法及免疫细胞化学染色检测目的蛋白表达 ,HE染色和电镜观察转染细胞的形态和结构的变化 ,细胞计数检测转染目的基因后细胞生长状况的变化 .结果 :成功地获得了人重构型caspase 6基因 (rcasp6 ) ,并构建了真核表达载体 .转染Hep 2细胞后 ,检测到目的蛋白的表达 .倒置显微镜观察转染细胞有明显变化 ,培养情况下出现大量细胞固缩变小 ,伴随有细胞死亡 .HE染色和电镜观察显示 ,固缩的细胞呈现凋亡的典型特征 .结论 :人重构型caspase 6基因在Hep 2细胞中表达 ,可使转染细胞的形态发生变化 。
AIM: To observe the effect of human reconstructed caspase 6 ( rcasp 6) gene on the transfected Hep 2 cell line and its expression in the Hep 2 cell line. METHODS: Human caspase 6 full length cDNA was obtained through RT PCR, and was reconstruted into reversed version of caspase 6 by settling the small subunit prior to the large one through PCR. The gene was cloned into vector pCMV and transfected into Hep 2 cells. Expression of rcasp6 protein was detected by immunofluorescent and immunocytochemical staining. Morphologic changes of Hep 2 cells subjected to transfection of rcasp6 gene were detected by HE staining and electron microscopy. RESULTS: Human reconstructed caspase 6 gene ( rcasp6 ) was cloned and inserted into pCMV vector. The Rcasp6 protein was detected in the cytoplasm of transfected Hep 2 cells. Many cells shrinked, and some cells were killed. Under electron microscope, those shrinked cells displayed typical features of apoptosis. CONCLUSION: Expression of human Rcasp6 protein in Hep 2 cells leads to apoptosis.
出处
《第四军医大学学报》
北大核心
2003年第24期2213-2216,共4页
Journal of the Fourth Military Medical University
基金
国家杰出青年科学基金 (3992 50 36)
军队杰出青年科学基金(98J0 0 9)
国家自然科学基金 (30 2 0 0 2 74)
国家高科技研究与发展计划资助 (2 0 0 1AA2 1 71 0 1 )
