CAJ | 학술논문

目的探讨抑制叉头状转录因子O1(FoxO1)基因表达对胰岛素抵抗HepG-2细胞葡萄糖消耗的影响及可能机制.方法构建针对转录因子FoxO1 mRNA特异性的携带红色荧光标记的siRNA载体,并测序鉴定.高浓度(10-6mol/L)胰岛素诱导24 h建立胰岛素抵抗细胞模型.实验共分4组:A组为普通培养基培养的细胞组;B组为未处理胰岛索抵抗细胞组;C组为转染FOxO1 siRNA载体的胰岛素抵抗细胞组;D组为以转染试剂为对照的胰岛素抵抗细胞组.转染后荧光显微镜下观察红色荧光的表达;葡萄糖氧化酶法检测各组细胞葡萄糖的消耗;RT-PCR技术检测FOxO1 mRNA表达;Western印迹和免疫沉淀技术检测胰岛素受体底物2(IRS-2)蛋白表达及酪氨酸磷酸化水平.结果经测序,成功构建了FoxO1 siRNA 载体.在转染后48 h,细胞内红色荧光表达最强.此时,与A组比较,B组葡萄糖消耗量降低(P<0.01),FoxO1 mRNA表达增强(P<0.05),IRS-2蛋白表达无显著性差别(P>0.05),但其酪氨酸磷酸化明显降低(P<O.01);与B组比较,C组FOxO1 mRNA表达明显降低(P<0.01),细胞葡萄糖消耗量增加(P<0.05),IRS-2蛋白酪氨酸磷酸化明显增强(P<0.01);与B组比较,D组各项指标表达差异不具有统计学意义(P>0.05).结论抑制FoxO1基因在胰岛素抵抗细胞中的高表达,可改善胰岛素敏感性,其机制可能是通过反馈调节IRS-2蛋白酪氨酸磷酸化,增强胰岛素信号转导.
목적 탐토억제차두상전록인자O1(FoxO1)기인표체대이도소저항HepG-2세포포도당소모적영향급가능궤제.방법 구건침대전록인자FoxO1 mRNA특이성적휴대홍색형광표기적siRNA재체,병측서감정.고농도(10-6mol/L)이도소유도24 h건립이도소저항세포모형.실험공분4조:A조위보통배양기배양적세포조;B조위미처리이도색저항세포조;C조위전염FOxO1 siRNA재체적이도소저항세포조;D조위이전염시제위대조적이도소저항세포조.전염후형광현미경하관찰홍색형광적표체;포도당양화매법검측각조세포포도당적소모;RT-PCR기술검측FOxO1 mRNA표체;Western인적화면역침정기술검측이도소수체저물2(IRS-2)단백표체급락안산린산화수평.결과 경측서,성공구건료FoxO1 siRNA 재체.재전염후48 h,세포내홍색형광표체최강.차시,여A조비교,B조포도당소모량강저(P<0.01),FoxO1 mRNA표체증강(P<0.05),IRS-2단백표체무현저성차별(P>0.05),단기락안산린산화명현강저(P<O.01);여B조비교,C조FOxO1 mRNA표체명현강저(P<0.01),세포포도당소모량증가(P<0.05),IRS-2단백락안산린산화명현증강(P<0.01);여B조비교,D조각항지표표체차이불구유통계학의의(P>0.05).결론 억제FoxO1기인재이도소저항세포중적고표체,가개선이도소민감성,기궤제가능시통과반궤조절IRS-2단백락안산린산화,증강이도소신호전도.
Objective To study the effects of inhibiting forkhead transcription factor O1 (FoxO1)expression by small interference RNA (siRNA) on the glucose utilization of insulin resistant HepG-2 cell line and the mechanism.Methods FoxO1 gene-targeted siRNA vector,which carried a red fluorescence,was constructed and then confirmed by DNA sequencing.HepG-2 cells were induced to a status of insulin resistance by being exposed to 10-6 mol/L insulin for 24 h.The study included 4 groups: HepG-2 cell group cultured with normal medium (group A) ; insulin resistant HepG-2 cell group (group B) ; insulin resistant HepG-2 cell group into which FoxO1 siRNA vector was transfected (group C) ; insulin resistant HepG-2 cell group into which Lipofectamine2000 was added (group D).The transfection efficiency could be estimated by observing the expression of red fluorescence.The expression of FoxO1 mRNA was analyzed by RT-PCR.The expressions of IRS-2 and tyrosine phosphorylation were detected by Western blot and immunoprecipitation.Results FoxO1 gene-targeted siRNA vector was built successfully.The expression of red fluorescence was the strongest at 48 h after transfection.Compared with group A,the glucose consumption and the expression of IRS-2 tyrosine phosphorylation of group B were decreased (P<0.01),and expression of FoxO1 mRNA was increased (P<0.05) in group B.There was no difference in the expressions of IRS-2 protein between A and B groups.Compared with group B,the expression of FoxOl mRNA was decreased (P < 0.01),and the glucose consumption and expression of IRS-2 tyrosine phosphorylation were significantly increased (P<0.05) in group C.There was no difference between group D and group B in glucose consumption, FoxO1 mRNA, IRS-2 protein and IRS-2 tyrosine phosphorylation expressions.Conclusions Inhibiting the expression of FoxO1 gene in insulin resistant HepG-2 cells seems to improve insulin sensitivity by feedback regulating the IRS-2 tyrosine phosphorylation expressions.