RASSF1A基因表达对肝癌细胞化疗药物敏感性影响.docVIP

RASSF1A基因表达对肝癌细胞化疗药物敏感性影响 　 作者：芮理，薛万江，李鹏，王鹏，王志伟，李厚祥 【摘要】 　　目的：研究RASSF1A提高肝癌细胞化疗药物敏感性的作用。方法：通过脂质体介导的基因转染法将RASSF1A基因的真核表达载体及空载体转染肝癌细胞株QGY-7703，G418筛选稳定表达株，并以RT-PCR和Western Blot进行鉴定目的基因的表达。化疗药物阿霉素(ADM)、丝裂霉素(MMC)、鬼臼乙叉甙(VP-16)、5-氟尿嘧啶(5-FU)、顺铂(DDP)分别作用各细胞株后，检测各细胞株的生长抑制率。结果：经800 μg/ml 的G418最佳筛选浓度筛选QGY-7703细胞株，成功建立稳定表达RASSF1A基因的肝癌细胞株。以QGY-7703细胞为对照，MMC对表达RASSF1A基因的肝癌细胞的生长抑制率最大为21%。结论：RASSF1A基因的表达可明显提高肝癌细胞对MMC的敏感性。RASSF1A的表达不影响肝癌细胞QGY-7703对ADM、 VP-16、5-FU和DDP敏感性。 【关键词】 肝细胞癌 Ras association domain family 1A 药物敏感性 逆转录聚合酶链反应 　　[Abstract] Objective：To investigate the effects of RASSF1A gene expression on the chemosensitivity of human hepatocellular carcinoma cell（HCC）. Methods：RASSF1A expression vector and empty vector were transfected into HCC cell line QGY-7703 with Lipofectamine 2000 reagents. The positive clones that expressed RASSF1A gene stably were chosen by G418 and checked by RT-PCR and Western Blot. Cell inhibited rate（IR） was measured after the treatment with the antitumor agents ADM, MMC, VP-16, 5-FU and DDP. Results：The HCC cell lines that reexpressed RASSF1A gene stably were established successfully. The suitable concentration of G418 used for selection agent was 800 μg/ml for QGY-7703 cell line. After being treated with MMC, the IR of cells reexpressing RASSF1A was maximal，and it was 21%, using QGY-7703 cell as control. Conclusion：　The expression of RASSF1A could increase the cell IR induced by MMC. RASSF1A gene expression had no effect on the chemosensitivity of human hepatocellular carcinoma cell treated with ADM, VP-16, 5-FU and DDP. 　　[Key words] Hepatocellular carcinoma; Ras association domain family 1A; Drug-sensitivity； Rerverse transcriptase polymerase chain reactionRASSF1A（ras association domain family 1A）基因是近年来发现的一个重要的抑癌基因，其抑制肺癌、肾癌、膀胱癌细胞生长的功能已得到证实[1~3]。通过瞬时转染发现，RASSF1A基因的表达使肺癌细胞株NCI-H1299生长阻止在G1/S期与抑制细胞周期蛋白cyclin D1的表达累积有关[4，5]。但对于RASS-F1A基因的表达对化疗药物敏感性的研究国内外尚未见报道，本研究通过建立稳定表达RASSF1A基因的肝癌细胞株，观察RASSF1A基因表达对肝癌细胞化疗药物敏感性的影响。 　　1 材料和方法 　　1．1 质粒、细胞株及主要试剂 pcDNA3