中文摘要：

为研究糖调节蛋白78（glucose regulated protein 78,GRP78）对肝细胞脂肪变性的影响,采用四氯化碳（carbon tetrachloride,CCl4）刺激人肝癌HepG2细胞,油红O染色证实,CCl4作用HepG2细胞后,细胞浆中脂肪颗粒明显增加,同时固醇调节元件结合蛋白1（sterol regulatory element bindingprotein 1,SREBP-1）蛋白水平和3羟3甲基戊二酸单酰CoA还原酶（HMGCoA还原酶）mRNA水平分别为对照组的1.55倍和1.70倍.构建人GRP78启动子荧光素酶报告基因载体pGL3/hGRP78P转染人肝癌HepG2细胞后,结果发现,CCl4促进GRP78基因转录,转录活性为诱导前的1.92倍.构建人GRP78 RNAi沉默质粒pSuper/GRP78转染人肝癌HepG2细胞后,该质粒能特异性沉默内源性GRP78;内源性GRP78沉默后的人肝癌HepG2细胞经CCl4诱导,HMGCoA还原酶mRNA和SREBP-1蛋白的表达较对照组进一步升高,分别为对照组的1.48倍和2.38倍;人肝癌HepG2细胞GRP78的体外过表达能降低CCl4介导的HMGCoA还原酶mRNA和SREBP-1蛋白诱导表达,分别为对照组的78.5%和51.5%;油红O染色进一步证实,GRP78过表达可明显减少脂肪颗粒在HepG2细胞浆中的集聚.综上表明,GRP78可抑制CCl4的SREBP-1和HMGCoA还原酶的诱导表达以及HepG2细胞脂肪变性,提示GRP78的表达增加在肝细胞脂肪变性损伤过程中具有潜在的保护作用.

英文摘要：

To elucidate the effect of glucose regulated protein 78（GRP78） on hepatic steatosis,CCl4-induced lipid deposition within HepG2 cells was adopted.The lipid accumulation in human HepG2 cells stimulated by CCl4 was confirmed by Oil Red O staining assay,and the levels of HMGCoA reductase mRNA and SREBP-1 protein in CCl4-treated HepG2 cells were increased to 1.55±0.07 folds（P〈0.05） and 1.70 folds of those in control group,respectively.The pGL3/hGRP78P reporter vector was constructed,in which the luciferase transcription was driven by human GRP78 promoter.In comparison with control group,the GRP78 transcriptional activity in CCl4-treated HepG2 cells was significantly enhanced to 1.92±0.09（P〈0.05） folds.The pSuper/GRP78 vector against endogenous GRP78 mRNA transcription was constructed and the specific GRP78 gene silencing was confirmed by quantitative RT-PCR.The CCl4-induced expression of HMGCoA reductase mRNA and SREBP-1 protein in those GRP78 gene-silencing HepG2 cells was enhanced by 48%±2%（P〈0.05） and 138% higher than that in control cells,whereas overexpression of GRP78 transfected by pcDNA3.1（＋）/hGRP78 plasmid could lead to inhibition of CCl4-induced up-regulation of HMGCoA reductase mRNA and SREBP-1 protein,down to 78.5%±0.58%（P〈0.05） and 51.5%,respectively.The overexpression of GRP78 also ameliorated CCl4-induced lipid accumulation within HepG2 cells.The gain and loss of function experiment in this research showed that CCl4-induced hepatic cell steatosis can be moderated by GRP78 protein,which is endoplasmic reticulum stress hallmark,and that GRP78 may be provided as a novel potential site for interfering with lipogenesis process in patients with hepatic steatosis.