目的 利用携带HCV复制子的Huh 7.5细胞探讨HCV复制对沉默信息调节因子1(SIRT1)表达和肝细胞葡萄糖代谢的影响.方法 应用流式细胞仪、比色法测定细胞活性氧(ROS)变化和烟酰胺腺嘌呤二核苷酸(NAD)+/还原型NDA(NADH)比值变化,应用液体闪烁计数仪、实时荧光定量-聚合酶链反应(RT-PCR)、Western印迹法检测SIRT1活性、mRNA和蛋白的表达,应用放射核素法和葡萄糖氧化酶法测定肝细胞葡萄糖摄取率和糖异生,RT-PCR检测SLRT1 下游调节糖代谢基因mRNA水平.计量资料比较采用t检验.结果 与Huh 7.5细胞相比,复制子细胞ROS水平升高(3.8±0.5比1.0±0.2,t=12.736,P＜0.01),NAD+/NADH比值下降(0.03±0.01比0.12±0.03,t＝6.971,P＜0.01),SIRT1活性(0.3±0.1比1.0±0.2,t＝7.668,P＜0.01)、mRNA(0.4±0.1比1.0±0.3,t=4.648,P＜0.01)和蛋白(0.3±0.1比0.8±0.2,t=5.941,P＜0.01)水平下降.SIRT1受抑后,不仅增加胰岛素受体底物-1(IRS-1)磷酸化(0.7±0.2比0.4±0.1,t=3.286,P＜0.01),降低蛋白激酶B(Akt)磷酸化(0.3±0.1比0.6±0.2,t=3.286,P＜0.01),下调葡萄糖转运蛋白2(GLUT2)基因表达(0.4±0.1比1.0±0.2,t=6.573,P＜0.01),抑制葡萄糖摄取率(每分钟计数值:4600±500比21 000±4600,t=8.682,P＜0.01)；而且降低叉头蛋白转录因子O1 (FoxO1)磷酸化(0.2±0.1比0.5±0.1,t=5.196,P＜0.01),上调磷酸烯醇丙酮酸羧激酶(2.8±0.6比1.0±0.3,t=6.573,P＜0.01)和葡萄糖-6-磷酸酶(2.6±0.5比1.0±0.2,t＝7.278,P＜0.01)基因表达,增加糖异生(2.5±0.5比1.0±0.2,t=5.543,P＜0.01).结论 HCV复制降低NAD+/NADH比值,下调SIRT1活性和表达,改变其下游调节糖代谢相关基因表达,降低葡萄糖摄取能力,增加糖异生,导致肝细胞葡萄糖代谢紊乱,促进HCV复制.
目的 利用攜帶HCV複製子的Huh 7.5細胞探討HCV複製對沉默信息調節因子1(SIRT1)錶達和肝細胞葡萄糖代謝的影響.方法 應用流式細胞儀、比色法測定細胞活性氧(ROS)變化和煙酰胺腺嘌呤二覈苷痠(NAD)+/還原型NDA(NADH)比值變化,應用液體閃爍計數儀、實時熒光定量-聚閤酶鏈反應(RT-PCR)、Western印跡法檢測SIRT1活性、mRNA和蛋白的錶達,應用放射覈素法和葡萄糖氧化酶法測定肝細胞葡萄糖攝取率和糖異生,RT-PCR檢測SLRT1 下遊調節糖代謝基因mRNA水平.計量資料比較採用t檢驗.結果 與Huh 7.5細胞相比,複製子細胞ROS水平升高(3.8±0.5比1.0±0.2,t=12.736,P＜0.01),NAD+/NADH比值下降(0.03±0.01比0.12±0.03,t＝6.971,P＜0.01),SIRT1活性(0.3±0.1比1.0±0.2,t＝7.668,P＜0.01)、mRNA(0.4±0.1比1.0±0.3,t=4.648,P＜0.01)和蛋白(0.3±0.1比0.8±0.2,t=5.941,P＜0.01)水平下降.SIRT1受抑後,不僅增加胰島素受體底物-1(IRS-1)燐痠化(0.7±0.2比0.4±0.1,t=3.286,P＜0.01),降低蛋白激酶B(Akt)燐痠化(0.3±0.1比0.6±0.2,t=3.286,P＜0.01),下調葡萄糖轉運蛋白2(GLUT2)基因錶達(0.4±0.1比1.0±0.2,t=6.573,P＜0.01),抑製葡萄糖攝取率(每分鐘計數值:4600±500比21 000±4600,t=8.682,P＜0.01)；而且降低扠頭蛋白轉錄因子O1 (FoxO1)燐痠化(0.2±0.1比0.5±0.1,t=5.196,P＜0.01),上調燐痠烯醇丙酮痠羧激酶(2.8±0.6比1.0±0.3,t=6.573,P＜0.01)和葡萄糖-6-燐痠酶(2.6±0.5比1.0±0.2,t＝7.278,P＜0.01)基因錶達,增加糖異生(2.5±0.5比1.0±0.2,t=5.543,P＜0.01).結論 HCV複製降低NAD+/NADH比值,下調SIRT1活性和錶達,改變其下遊調節糖代謝相關基因錶達,降低葡萄糖攝取能力,增加糖異生,導緻肝細胞葡萄糖代謝紊亂,促進HCV複製.
목적 이용휴대HCV복제자적Huh 7.5세포탐토HCV복제대침묵신식조절인자1(SIRT1)표체화간세포포도당대사적영향.방법 응용류식세포의、비색법측정세포활성양(ROS)변화화연선알선표령이핵감산(NAD)+/환원형NDA(NADH)비치변화,응용액체섬삭계수의、실시형광정량-취합매련반응(RT-PCR)、Western인적법검측SIRT1활성、mRNA화단백적표체,응용방사핵소법화포도당양화매법측정간세포포도당섭취솔화당이생,RT-PCR검측SLRT1 하유조절당대사기인mRNA수평.계량자료비교채용t검험.결과 여Huh 7.5세포상비,복제자세포ROS수평승고(3.8±0.5비1.0±0.2,t=12.736,P＜0.01),NAD+/NADH비치하강(0.03±0.01비0.12±0.03,t＝6.971,P＜0.01),SIRT1활성(0.3±0.1비1.0±0.2,t＝7.668,P＜0.01)、mRNA(0.4±0.1비1.0±0.3,t=4.648,P＜0.01)화단백(0.3±0.1비0.8±0.2,t=5.941,P＜0.01)수평하강.SIRT1수억후,불부증가이도소수체저물-1(IRS-1)린산화(0.7±0.2비0.4±0.1,t=3.286,P＜0.01),강저단백격매B(Akt)린산화(0.3±0.1비0.6±0.2,t=3.286,P＜0.01),하조포도당전운단백2(GLUT2)기인표체(0.4±0.1비1.0±0.2,t=6.573,P＜0.01),억제포도당섭취솔(매분종계수치:4600±500비21 000±4600,t=8.682,P＜0.01)；이차강저차두단백전록인자O1 (FoxO1)린산화(0.2±0.1비0.5±0.1,t=5.196,P＜0.01),상조린산희순병동산최격매(2.8±0.6비1.0±0.3,t=6.573,P＜0.01)화포도당-6-린산매(2.6±0.5비1.0±0.2,t＝7.278,P＜0.01)기인표체,증가당이생(2.5±0.5비1.0±0.2,t=5.543,P＜0.01).결론 HCV복제강저NAD+/NADH비치,하조SIRT1활성화표체,개변기하유조절당대사상관기인표체,강저포도당섭취능력,증가당이생,도치간세포포도당대사문란,촉진HCV복제.
Objective The aim of this study was to investigate the effect of hepatitis C virus (HCV) replication on expression of silent information regulator 1 (SIRT1) and glucose metabolism of hepatocytes using Huh 7.5 cells harboring HCV replicon.Methods The level of reactive oxygen species (ROS),value of nicotinamide adenine dinucleotide (NAD+)/reduced form of nicotinamide adenine dinucleotide (NADH) was detected by flow cytometry and chromatometry.The activity,mRNA expression,and protein level of SIRT1 were detected by a scintillation counter,real-time fluorescence quantitative polymerase chain reaction (RT-PCR),and Western blot,respectively.Glucose uptake by hepatocytes and gluconeogenesis were detected using radioactive isotope method and glucose oxidase method.The mRNA levels of SIRT1 downstream glucose-metabolism genes were measured by RT-PCR.Measurement date were compared by t test.Results In replicon cells,the level of ROS (3.8±0.5 vs 1.0±0.2; t=12.736,P＜0.01) was increased and the value of NAD+/NADH (0.03±0.01 vs 0.12±0.03; t=6.971,P＜0.01) decreased compared with Huh 7.5 cells.The activity (0.3±0.1 vs 1.0±0.2; t=7.668,P＜0.01),mRNA expression(0.4±0.1 vs 1.0± 0.3; t=4.648,P＜0.01) and protein level(0.3±0.1 vs 0.8±0.2; t=5.941,P＜0.01) of SIRT1 were reduced.Inhibition of SIRT1 not only increased insulin receptor substrate-1 (IRS-1) phosphorylation (0.7±0.2 vs 0.4±0.1; t=3.286,P＜0.01),decreased protein kinase B (Akt) phosphorylation (0.3 ± 0.1 vs 0.6 ± 0.2; t=3.286,P＜0.01),down regulated cell surface expression of glucose transporler 2 (GLUT2,0.4±0.1 vs 1.0 ± 0.2; t =6.573,P＜0.01) and suppressed cellular glucose uptake (count per minute:4600±500 vs 21 000±4600; t=8.682,P＜0.01); but also decreased phosphorylation of forkhead box O1 (FoxO1,0.2＝0.1 vs 0.5±0.1; t=5.196,P＜ 0.01),up-regulated phosphoenolpyruvate carboxykinase (PEPCK,2.8±0.6 vs 1.0±0.3; t=6.573,P＜0.01) and glucose 6-phosphatase (2.6±0.5 vs 1.0±0.2; t=7.278,P＜0.01) genes,and promoted glucose production (2.5±0.5 vs 1.0±0.2; t=5.543,P＜0.01).Conclusions HCV replication decreases NAD+/NADH ratio,which might down-regulate the activity and the expression of SIRT1,leading to changes in the expression profile of glucose metabolism related genes and causing glucose metabolism disorders of hepatocytes by a decrease in glucose uptake and an increase in glucose production,and promotes HCV replication.
