COX-2 siRNA诱导人肝癌Hep3B细胞凋亡实验研究

THE INDUCTION OF COX-2 SIRNA ON PROLIFERATION AND APOPTOSIS OF HEP3B HEPATOCARCINOMA CELLS

目的:观察COX-2在人肝癌细胞Hep3B中的表达及小干扰RNA(small interfering RNA,siRNA)沉默COX-2基因对COX-2 mRNA和蛋白表达、细胞增殖及凋亡的影响。方法:将siRNA-COX-2质粒载体转染Hep3B细胞,WST-8法检测细胞增殖抑制情况,透射电镜观察凋亡小体出现情况;筛选单克隆细胞株Hep3B-sh,采用RT-PCR和Western blotting检测COX-2 mRNA和蛋白情况。结果:siRNA-COX-2能抑制Hep3B细胞的增殖,稳定转染pshRNA-COX-2可使Hep3B细胞中COX-2 mRNA及蛋白的表达明显减弱(P<0.01);透射电镜下可见凋亡小体出现,发生凋亡细胞数增加。结论:COX-2siRNA能有效抑制肝癌Hep3B细胞中COX-2的表达和细胞的增殖,促进凋亡发生,为肝细胞癌基因治疗提供实验依据和治疗靶点。

Objective： To investigate the influence of siRNA targeted COX-2 gene in Hep3B cells on prolif- eration, apoptosis and COX-2 mRNA and protein expressions in malignant hepatoma carcinoma cell line Hep3B after siRNA stably inhibited cell lines. Methods： Hepatocelluar carcinoma cell Hep3B were transfected by Plas- raids siRNA. Stably inhibited COX-2 siRNA cell lines of Hep3B were established. The effect of siRNA COX-2on Hep3B cells＇ proliferation was assayed by WST-8. Transmission electron microscope was used to examine the apoptosis effects on ceils. Reverse transcription-polymerase chain reaction （RT-PCR） and western blotting were applied to detect the mRNA and protein expression of COX-2 in Hep3B cells of three groups respectively. Re- sults： Recombinant expression plasmid siRNA-COX-2 could significantly inhibit Hep3B cells proliferation underthe condition of stably expression. In Hep3B-sh cells, mRNA level in cells and the production of COX-2 was microscope showed that large number of apoptotic bodies the expression of COX-2 was significantly attenuated at significantly decreased （P〈0.01）,Transmission electron appeared in Hep3B-sh cells . Conclusion：Recombinantexpression vector siRNA-COX-2 can significantly inhibit malignant hepatoma carcinoma cell proliferation and the expression of COX-2 gene and protein and promote apoptosis. It suggests that COX-2 gene may become a new target in gene therapy. These experiments in vitro can provide experimental evidence for gene therapy.