Raf激酶抑制蛋白对HepG2肝癌细胞生物学特性影响的研究

摘要

Objective: RKIP is one of the most important members of the PEBP family. The results of recent researches suggested that the expression of this gene could decrease in human liver cancer cells. The aim of the research was to investigate the influence of RKIP on the growth, proliferation, apopatosis, invasionn and cell cycle of the liver cancer liner HepG2. Methods: The cDNA of RKIP was subcloned into a constitutive vector pcDNA3. 1 followed by transfection in HepG2 cell by using liposome. Then stable expression clones were selected and renamed as Hep -RKIP. The apoptosis and cell cycles were detected using flow cytometry. The growth and proliferation were analyzed by making cell growth curves and colony formation assay respectively. The ability of invasion were tested using cancer cell migration assay. Results: Hep - RKIP group grew slower than HepG2 and Hep - PC. The cell counts of Hep - RKIP in four of seven days were fewer than those of control groups significantly( P < 0. 05 ). Cell cycle analysis showed that proportions of Hep - RKIP cells in Go - G, were higher and G2 - M were lower significantly( P <0.05 ) than those of two control groups. The results of colony formation assay showed that the colony formation rate of Hep - RKIP was lower than those of control groups( P < 0. 05 ). The results of cell migration assay suggested that the cell migration rate of Hep - RKIP was lower significantly that those of control groups( P < 0.05 ). Conclusion: RKIP can restrain the growth or proliferation of HepG2 cell and decrease the proportion of cells in division stage. It can restrain invasion and metastasis of liver cancer cells. So RKIP gene can reduce the maintain malignant phenotypes of liver cancer cell.%目的 Raf激酶抑制蛋白(RKIP)是磷脂酰乙醇胺结合蛋白家族的重要成员,近来的一些研究表明RKIP基因的蛋白产物在人类肝癌组织中表达显著下调.本研究旨在探讨RKIP基因对于肝癌细胞增殖状态,细胞周期,凋亡状态等生物学特性及浸润转移能力等恶性表型的影响,以初步阐明其功能意义.方法 将RKIP基因插入载体pcDNA3.1构建PC-RKIP真核表达载体.脂质体转染肝癌细胞系HepG2建立稳定转染细胞系(Hep-RKIP),而后使用生长曲线法、平板克隆形成实验法、流式细胞仪,细胞迁徙实验法等方法分析稳定表达株相关生物学特性变化.同时设立空载体转染组和空白细胞组为对照.结果 与转染pcDNA3.1空载体及空白HepG2肝癌细胞相比,转染PC- RKIP载体的稳定表达细胞株生长有显著减慢,后两组之间亦无显著差异,平板克隆形成实验结果显示PC- RKIP转染组平均克隆形成率显著低于其它两组(P<0.05),细胞周期检测显示PC- RKIP转染组处于G0/G1期的细胞比例显著高于未处理的HepG2 细胞和转染 pcDNA 3.1空载体的HepG2细胞(P<0.05),而处于G2-M期的细胞比例显著均低于其它两组(P<0.05),其它各期细胞比例均无显著差异.流式细胞仪法检测细胞凋亡结果显示PC-RKIP转染组调亡比例为6.4%左右,显著高于对照组(P<0.05).细胞迁徙实验结果提示PC-RKIP转染组穿膜率与其它两组相比显著降低(P<0.05).结论 RKIP基因可能具有抑制细胞生长增殖及分裂作用,同时可影响细胞周期及细胞凋亡,对肝癌细胞侵袭转移能力可能有一定抑制作用,对抑制细胞恶性表型有一定意义.