乙型肝炎表面抗原主蛋白对HepG2细胞脂代谢基因表达的影响

Hepatitis B surface antigen affects the expression of lipid metabolism-related genes in HepG2 cells

目的研究HBsAg主蛋白对肝细胞中脂代谢基因表达的影响。方法将PCR扩增C型HBV主s基因（SHIN）定向克隆至pcDNA3．1（＋）载体中，重组质粒pcDNA3．1-SHBs与pcDNA3．1分别转染HepG2细胞，筛选出稳定转染细胞株。用实时定量PCR和Westernb10t法检测两细胞株中脂代谢相关基因mRNA及蛋白水平表达差异。结果成功建立稳定表达SHIN的细胞株HepG2-pn3．1-SHIN及空质粒对照细胞株HepG2-pn3．1-neo。实时定量PeR检测相关基因表达差异结果显示：HepG2-pn3．1-SHIN细胞组中基因烯酰辅酶A水合酶、载脂蛋白A、脂蛋白脂酶mRNA相对表达水平较低，分别与HepG2-pn3．1-neo细胞组比较（0．161±0．043对比0．210±0．022，t=2．479；0．031±0．007对比0．094土0．055，t=2．752；0．770±0．036对比0．982±0．031，t=10．914），P值均〈0．05，差异均有统计学意义;而HepG2-pn3．1-SHIN细胞组基因乙酰辅酶A羧化酶、胆固醇调节元件结合蛋白-1cmRNA相对表达水平较高，分别与HepG2-pn3．1-neo细胞组比较（0．113士0．027对比0．059±0．022，t=-3．757；0．019±0．002对比0．015±0．001，t=-4．330），P值均〈0．05，差异均有统计学意义。两组肉毒碱脂酰辅酶A转移酶-1、过氧化物酶体增生物激活受体α的基因转录水平比较，差异虽无统计学意义，但呈减弱趋势。Westernblot结果显示上述基因蛋白水平的变化趋势与mRNA水平一致。结论SHBs能改变脂肪酸合成，分解相关基因的表达，但HBsAg是否直接导致肝细胞脂肪变性尚不明确，值得进一步研究。

Objective To investigate the influence of hepatitis B virus （HBV）-encoded small surface protein （SHBs） on hepatic cell expression of host genes related to lipid metabolism. Methods The full-length SHBs gene was amplified from HBV genotype C by polymerase chain reaction （PCR） and cloned into the pcDNA3.1（＋） expression vector for stable transfection into HepG2 cells （selected by G418 screening）; cells transfected with empty vector served as control. The SHBs mRNA and protein levels were detected by reverse transcription-PCR and enzyme-linked immunosorbent assay. SHBs effects on expression of genes and proteins related to lipid metabolism were detected by real-time quantitative （q） PCR and western blotting, respectively. Results The stably transfected cell line HepG2-pn3.1-SHBs was established successfully, qPCR showed that the HepG2-pn3.1-SHBs cells had significantly down- regulated transcription of the ECHS1, APOA1 and LPL genes （0.161 q- 0.043 vs. control cells： 0.210 ±0.022, t = 2.479; 0.031 ± 0.007 vs. 0.094 ± 0.055, t = 2.752; 0.770 ± 0.036 vs. 0.982 ＋ 0.031, t = 10.914）, but significantly up-regulated ACC and SREBP-lc genes （0.113 ± 0.027 vs. 0.059 ±0.022, t = -3.757; 0.019 ± 0.002 vs. 0.015 ± 0.001, t = -4.330）. The CPTla and PPARa genes＇ expression was slightly, but not significantly, down-regulated in the HepG2-pn3.1-SHBs cells （0.028 ±0.005 vs. 0.030 ± 0.004, t = 1.022; 0.014 ± 0.004 vs. 0.015 ±0.002, t = 0.758）. Western blotting showed similar expression trends for the corresponding proteins. Conelusion SHBs alters the expression of some host genes with known functions in fatty acid synthesis and decomposition; however, it remains unclear whether the hepatitis B surface antigen can directly contribute to development of hepatic steatosis.