脂肪变性肝细胞胆固醇代谢及合成相关基因的表达

Cholesterol metabolism and its relevant genes expression in cultured steatotic hepatocytes

目的探讨脂肪变性肝细胞胆固醇代谢及合成相关基因mRNA表达的变化。方法以软脂酸诱导正常成人肝细胞株L-02脂肪变性,建立肝细胞脂肪变性模型,分别于实验第3、6天收集细胞,同期设不含软脂酸培养的细胞作对照。试剂盒检测细胞内甘油三酯(TG)和总胆固醇(TC)含量,RT-PCR法检测固醇调节元件结合蛋白2(SREBP-2)及其靶基因羟甲基戊二酸单酰辅酶A还原酶(HMGCR)、低密度脂蛋白受体(LDLR)mRNA表达。结果软脂酸诱导第3天即可产生肝细胞脂肪变性,第6天脂肪变性加重。随造模时间延长,模型组细胞内TG、TC含量逐渐增多,SREBP-2、HMGCR、LDLR mRNA表达逐渐增强;第3天和第6天细胞内TG含量均显著高于对照组(P<0.05),第6天细胞内TC含量显著高于对照组(P<0.05);第3天和第6天HMGCR、LDLR mRNA表达显著高于对照组(P<0.05);第6天SREBP-2 mRNA表达显著高于对照组(P<0.05)。结论脂肪变性肝细胞内存在胆固醇积聚,其机制可能是胆固醇合成相关基因表达上调。

Objective To explore the cholesterol metabolism and expression of mRNA of relevant genes in cholesterol synthesis in the models of cultured steatotic hepatocytes. Methods Steatosis model of hepatocytes was established by adding palmitic acid to the growing L-02 cells. The cells were collected at day 3 and 6, respectively. The cells added culture solution without palmitic acid were served as control. The intracellular triglyceride（TG） and total cholesterol（TC） were detected by analyzed kit. The expressions of sterol-regulatory element binding protein-2 （SREBP-2） and its target gene hydroxymethylglutaryl CoA reductase （HMGCR） and low density lipoprotein receptor （LDLR） were measured with RT-PCR. Results Hepatocyte steatosis was observed at day 3, and became more serious at day 6. The contents of intracellular TG and TC were increased, and the expressions of SREBP-2, HMGCR and LDLR mRNA were upregulated in a time-dependent manner in the model group. Compared with the control group, the content of intracellular TG was higher both at day 3 and 6 （ P 〈 0.05） , while the content of intracellular TC was significantly increased only at day 6. The expression of HMGCR and LDLR mRNA was up-regulated in steatotic hepatocytes both at day 3 and 6 （P 〈0.05）, while the SREBP-2 mRNA was increased only at day 6 （ P 〈 0.05）. Conclusion There is a cholesterol accumulation as a result of increased expression of relevant genes in cholesterol synthesis in steatotic hepatocytes.