中文摘要：

目的合成和筛选有效抑制细胞内氯离子通道蛋白1（CLIC1）基因的shRNA序列，构建表达质粒，并进一步研究CLIC1基因的表达被抑制后小鼠肝癌细胞株Hca-F细胞增殖及侵袭能力的变化。方法设计并合成3条理论上最佳的siRNA序列，进而将相应的双链DNA插入pGPU6／GFP／Neo质粒中，以脂质体Lipofectamine2000将DNA质粒稳定转染至小鼠Hca-F细胞中，收取细胞进行逆转录多聚酶链反应分析各组的CLIC1 mRNA表达水平，选出最佳干扰序列。应用细胞计数试剂盒检测最佳干扰序列表达质粒稳定转染的Hca-F细胞，对比观察其干扰前后增殖情况的变化；应用transwell小室检测其侵袭能力的变化。对研究数据应用统计学软件SPSS13．0进行方差分析。结果靶向CLIC1 mRNA的3个shRNA重组质粒载体经测序分析，shRNA编码序列与设计的片段完全一致。经酶切凝胶电泳证实载体构建成功。稳定转染pGPU6／GFP／Neo-shRNA-3表达质粒的Hca-F细胞对其CLIC1 mRNA抑制效果明显，抑制率达42．4％。经该表达质粒的干扰后，Hca—F细胞的增殖能力明显增强，侵袭能力显著下降，空白对照组、无关序列处理组、shRNA干扰组的穿膜细胞个数分别为98．93±5．00、96．27±2．60、50．73±3．89，P值均〈0．01。结论pGPU6／GFP／Neo-shRNA-3表达质粒能高效地抑制小鼠Hca-F细胞中CLIC1 mRNA的表达，CLIC1基因具有抑制小鼠肝癌细胞的增殖和促进其侵袭的作用。

英文摘要：

Objective To study the effects of silencing CLIC1 gene expression on the proliferation and invasion of Hca-F cells. Methods The mouse CLIC1 cDNA sequence was retrieved from NCBI. Three shRNA sequences were designed and cloned into pGPU6/GFP/Neo plasmids. The plasmids were transfected into Hca-F cells with Lipofectamine 2000. Cell Counting-8 （CCK-8） kit and transwell chamber were used to study the effects of CLIC1 on the proliferation and invasion of Hca-F cells. Results The pGPU6/GFP/Neo- shRNA-3 plasmid effectively repressed the expression of CLIC1 mRNA. Inhibition of CLIC1 gene expression led to decreased cell proliferation and reduced invasion. Conclusion CLIC1 is essential for the proliferation and invasion of Hca-F cells.