PNPLA3在实验性非酒精性脂肪性肝病中的表达及其CpG岛甲基化状态的研究

The Expression and CpG Island Methylation of PNPLA3 in Experimental Nonalcoholic Fatty Liver Disease

目的:探讨PNPLA3在非酒精性脂肪性肝病中的表达水平及其Cp G岛甲基化状态,探讨PNPLA3在NAFLD发生、发展中的作用。方法:1.采用10μg/m L的油酸作用于人正常肝细胞株L02建立脂肪肝细胞模型,72 h后经油红O染色,观察正常组、脂肪肝模型组及给药组细胞内脂滴形成情况,并用荧光定量PCR检测PNPLA3在三组肝细胞中的表达情况。2.采用专用DNA提取试剂盒分别提取正常组、脂肪肝模型组、给药组细胞中DNA,经亚硫酸转化后行PCR扩增目的基因,并用焦磷酸测序方法检测不同组PNPLA3 Cp G岛中甲基化水平,探讨其在非酒精性脂肪肝中的作用。结果:1、脂肪肝模型组肝细胞PNPLA3 m RNA的表达高于正常组,经姜黄素给药后,其表达降低,差异均有统计学意义(P<0.05)。2、在PNPLA3启动子区6个检测位点中,脂肪肝组位点2甲基化水平高于正常组,姜黄素给药后甲基化水平降低,差异均有统计学意义(P<0.05),在其余各检测位点中甲基化水平无差异。结论:1、油红O染色后,脂肪肝模型组细胞内发现有大量红色脂滴积聚,而正常组基本上未见脂滴,姜黄素给药组脂滴较模型组减少,表明用10μg/m L的油酸诱导能成功建立体外肝细胞模型。2、PNPLA3 m RNA在肝细胞中高表达及其启动子区甲基化状态异常参与非酒精性脂肪肝发病。3、抑制肝脏PNPLA3活性或降低肝细胞PNPLA3 m RNA的表达水平可能是今后治疗非酒精性脂肪性肝病(NAFLD)的一个新的治疗方法。

Objective：To investigate the expression and CpG island methylation of PNPLA3 in experimental nonalcoholic fatty liver disease,and explore the role of gene PNPLA3 in development of NAFLD.Methods：The normal L02 hepatocytes were treated with 10 μg/mL oleic acid to establish nonalcoholic fatty liver disease （NAFLD） cell models.Quantitative real-time PCR was used to analyze PNPLA3 gene expression in control group,NAFLD group and curcumin group （treated with10.0 μM curcumin）.The DNA methylation level was analyzed by pyrosequencing.Results：The expression level of PNPLA3 mRNA in NAFLD group was much higher than the control group.After curcumin treatment,PNPLA3 mRNA expression decreased when compared with the NAFLD group.The methylation level at point 2 of the PNPLA3 gene promoter region in NAFLD group was higher than that in the normal group.Conclusion：1.After oil red O staining,much more red lipid droplets accumulate in cytoplasm of NAFLD group than control group,indicating that 10 μg/mL oleic acid can successfully induce nonalcoholic hepatocellular steatosis.2.The higher level of expression and abnormal methylation of PNPLA3 were correlated with NAFLD.3.Inhibition of the hepatic PNPLA3 activity and decreasing the expression of PNPLA3 may represent a novel therapeutic approach for the treatment of NAFLD.