肝再生增强因子,细胞色素P450与人肝癌细胞在体外耐药的关系

The relationship between augmenter of liver regeneration, cytochrome P450 and the drug resistance of induced human hepatocellular carcinoma drug-resistant cells in vitro

目的:研究肝癌顺铂耐药细胞HepG2/cDDP中人肝再生增强因子(Human augmenter of liver regeneration,hALR)对细胞色素P450(Cytochrome P450,CYP450)表达的影响,观察ALR是否通过作用CYP450与肝癌细胞耐药的形成有关。方法:建立体外肝癌顺铂耐药细胞系,MTT检测耐药指数;将构建好的人肝再生增强因子siRNA表达质粒psiALR-A及其阴性对照质粒psiALR-B采用脂质体转染的方法分别转染至HepG2/cDDP细胞,荧光显微镜观察绿色荧光蛋白的表达,计算转染效率。根据转染不同的质粒,将HepG2/cDDP细胞分为3组:转染组(转染psiALR-A),对照组(转染psiALR-B),空白组(未转染重组质粒)。免疫细胞化学法、免疫印迹法、实时荧光定量PCR法检测转染细胞中hALR和CYP450的表达,化学发光法检测各组细胞中CYP3A4的活性,MTT比色法检测各组细胞对顺铂敏感性的变化。结果:耐药模型成功建立,耐药指数为6.26;成功转染质粒psiALR-A和psiALR-B;转染组细胞中hALR表达明显减少,CYP450的表达明显增加,活性升高,而对照组和空白组细胞中hALR和CYP450表达没有明显的变化。MTT结果显示转染组细胞对顺铂的敏感性降低,对照组和空白组细胞对顺铂的敏感性没有明显变化。结论:阻断人肝再生增强因子的表达后,HepG2/cDDP细胞CYP450表达明显增加,活性增强,对顺铂的敏感性下降,ALR可能通过作用CYP450在肝癌细胞耐药的发生发展中起着重要作用。

Objective：To investigate the effects of augmenter of liver regeneration（hALR） in cisplatin（cDDP） induced human hepatocellular carcinoma drug-resistant cells（HepG2/cDDP） on the expression of cytochrome P450,and detect whether ALR has relationship with the drug resistance of human hepatocellular carcinoma cells by affecting CYP540.Methods： A drug resistant human hepatoma cell line（HepG2/cDDP） induced by cisplatin was established in vitro,and its resistance index was evaluated by MTT assay.The expressing siRNA plasmid psiALR-A targeting hALR and the unrelated control plasmid psiALR-B were constructed successfully and transfected into HepG2/cDDP cells with lipofectamine 2000 methods,respectively;the expression of green fluorescent protein was observed under a fluorescence microscope to calculate transfection efficiency.According to different plasmid transfection,HepG2/cDDP cells were divided into three groups： transfection group（transfected psiALR-A）,control group（transfected psiALR-B）,and blank control group（non-transfected with the recombinant plasmid）.The protein and mRNA level of hALR and CYP450 in each group of cells were measured by immunocytochemistry,Western Blot and Real-time FQ-PCR.The activities of CYP450 in each group of cells were detected by chemiluminescence;drug sensitivity to cDDP in each group cells was detected by MTT assay.Results： HepG2/cDDP cell line was established successfully with stable resistance to cDDP,and the resistance index（RI） was 6.26.Plasmid psiALR-A targeting hALR and the unrelated control plasmid psiALR-B were transfected successfully.In transfection group,the level of hALR protein and mRNA expression decreased significantly,but the level of CYP450 protein and mRNA expression increased significantly.In the control group and blank control group,there was no obvious change.The activities of CYP450 were increased significantly and the sensitivity to cDDP was decreased in transfection group;however,there was no obvious change in the control group and blank control group.Conclusion： The level of CYP450 expression and the activities of CYP450 are increased significantly,but the sensitivity to cDDP is decreased in transfection group.ALR may play an important role in the occurrence and development of the drug resistance of hepatocellular carcinoma cells.