摘要：
目的 研究丙型肝炎病毒(HCV)核心蛋白对肝窦内皮细胞(LSEC)沉默信息调节因子1(SIRT1)表达及LSEC功能的影响.方法 HepG2细胞或表达HCV核心蛋白的HepG2细胞与LSEC共培养.应用液体闪烁计数仪、实时荧光定量-PCR(RT-PCR)、Western印迹检测LSEC SIRT1活性、mRNA和蛋白的表达,以及LSEC脂联素受体2(AdipoR2)、内皮型一氧化氮合酶(eNOS)、第Ⅷ相关抗原(Von Willebrand factor, vWf)、分化抗原簇31(CD31)、血管内皮细胞生长因子(VEGF)和CD14蛋白的表达.应用流式细胞仪检测细胞活性氧(ROS)水平,检测共培养上清液中丙二醛(MDA) 、超氧化物歧化酶(SOD)、脂联素、一氧化氮(NO)和内皮素-1(ET-1)的水平.计量资料采用t检验.结果 与LSEC和HepG2细胞共培养组相比,LSEC和表达HCV核心蛋白HepG2细胞共培养组SIRT1活性(0.3±0.1比1.0±0.3,t=5.422,P<0.01)、mRNA(0.4±0.1比1.0±0.2,t=6.573,P<0.01)和蛋白(0.3±0.08比1.0±0.3,t=5.613,P<0.01)水平下降;脂联素[(3.41±0.61)比(5.82±0.87)μg/mL,t=5.556,P<0.01]分泌减少且AdipoR2蛋白(0.3±0.1比0.8±0.2,t=5.477,P<0.01)表达下降;eNOS蛋白(0.4±0.1比0.9±0.3,t=3.873,P<0.01)表达下降;vWf蛋白(0.8±0.3比0.4±0.1,t=3.098,P<0.01)、CD31蛋白(0.9±0.2比0.3±0.1,t=6.573,P<0.01)、VEGF蛋白(0.9±0.3比0.5±0.1,t=3.873,P<0.01)表达增加;CD14蛋白(0.4±0.1比0.9±0.3,t=3.873,P<0.01)表达下降.共培养上清液中NO和SOD水平下降,ET-1和MDA水平升高. 结论 HCV核心蛋白下调SIRT1活性及表达,下调脂联素及受体表达,引起LSEC收缩和肝窦毛细血管化,增加氧化应激反应,导致肝窦微循环障碍.%Objective To investigate the effect of hepatitis C virus (HCV) core protein on expression of silent information regulator 1 (SIRT1) and function of liver sinusoidal endothelial cell (LSEC).Methods LSEC was co-cultured with HepG2 cells with/without expressing HCV core protein, respectively.After co-culture, the activity and expression levels of SIRT1 in LSEC were detected by scintillation counter in mRNA and protein levels, using real-time polymerase chain reaction (RT-PCR) and western blot, respectively.Levels of adiponectin receptor 2 (AdipoR2), endothelial nitric oxide synthase (eNOS), von Willebrand factor (vWf), cluster of differentiation (CD) 31, vascular endothelial growth factor (VEGF) and CD14 were measured using western blot.Level of reactive oxygen species (ROS) was assayed using flow cytometry.Levels of malondialdehyde (MDA), superoxide dismutase (SOD), adiponectin, nitric oxide (NO) and endothelin-1 (ET-1) in the supernatant were measured.The quantitative data was analyzed using t-test.Results Compared with co-culture cells without expressing HCV core protein, the co-culture cells with expressing HCV core protein showed lower activity (0.3±0.1 vs 1.0±0.3, t=5.422, P<0.01), mRNA (0.4±0.1 vs 1.0±0.2, t=6.573, P<0.01) and protein (0.3±0.08 vs 1.0±0.3, t=5.613, P<0.01) of SIRT1;vWf protein (0.8±0.3 vs 0.4±0.1, t=3.098, P<0.01), CD31 protein (0.9±0.2 vs 0.3±0.1, t=6.573, P<0.01) and VEGF protein (0.9±0.3 vs 0.5±0.1, t=3.873, P<0.01);lower levels of adiponectin (3.41±0.61 vs 5.82±0.87μg/mL, t=5.556, P<0.01), AdipoR2 protein (0.3±0.1 vs 0.8±0.2, t=5.477, P<0.01), eNOS protein (0.4±0.1 vs 0.9±0.3, t=3.873, P<0.01) and CD14 protein (0.4±0.1 vs 0.9±0.3, t=3.873, P<0.01), respectively.Additionally, compared to those in the supernatant of co-culture cells without expressing HCV core protein, NO and SOD levels were decreased in co-culture cells with expressing HCV core protein, whereas ET-1 and MDA levels were increased.Conclusion HCV core protein may down-regulate the activity and expression of SIRT1, decrease adiponectin and AdipoR2, subsequently induce LSEC contraction and hepatic sinusoidal capillarization, as well as increase oxidative stress, ultimately lead to liver microcirculation disturbance.
