COX-2反义RNA抑制肝癌细胞增殖的实验研究

Effects of liposome-mediated rat cyclooxygenase-2 antisense RNA on growth of hepatocarcinoma cells

目的研究COX-2反义RNA对肝癌细胞增殖抑制的作用及其机制，探讨肝癌治疗的新途径。方法人工合成的COX-2反义RNA片段及无关对照空质粒经脂质体包裹后作用CBRH7919细胞，通过MTT法、细胞周期分析、RT—PCR及裸鼠体内接种等方法测定细胞体内外增殖的变化。结果经COX-2反义RNA处理的CBRH7919细胞与对照组CBRH7919细胞相比，体外增殖速度减慢（细胞增殖抑制率达78％）、DNA合成受抑（S期细胞数为34．8％VS59．9％），细胞周期G0／G1比例明显提高，裸鼠体内成瘤率下降（25％ vs 100％）。而凋亡相关基因表达并无明显差异（P〉0．05）。结论COX-2反义RNA可有效抑制肝癌细胞的体内外生长增殖，可用于实验性肿瘤基因治疗研究。

Objective To construct a recombinant eukaryotic expression vector encoding rat COX-2 antisense RNA and investigate its effects on rat liver cancer cell proliferatiion. Methods The plasmid encoding anti-sense COX-2 was constructed by using cloning COX-2 cDNA fragment in the re- verse direction into the pcDNA3. 1. Then the plasmid pcDNA3. 1/COX-2as was transfered into rat hepatocacinoma cell line CBRH7919 with liposome and homologous recombination cell was named as CBRH7919-A. The cell proliferation, cell cycle and apoptosis were analyzed by MTT, flow cytometry and RT-PCR. CBRH7919 cells with or without pretreatment were inoculated into nude mice and the cell proliferation was observed in vivo. Results CBRH7919 treated with antisense COX-2 greatly in- hibited the proliferation with a rate of 78% and DNA synthesis （PI of COX-2 group vs control group, 0. 361 vs 0. 784） of CBRH7919 cell line, decreased coloning formation in anchorage-independent as- say. In vivo tumorigenic rate was greatly reduced in athymic nude mice as compared with untreated cell group （25% vs 100%）. Apoptosis-related gene expression was not different as compared with un treated cell group （P〉0.05）. Conclusion Antisense COX-2 RNA can inhibit the proliferation of rat liver cancer cell line in vitro as well as in vivo, suggesting that it has potential value in hepatocellular cancer gene therapy.