SIRT1 siRNA对胰岛素抵抗模型小鼠脂代谢和血小板功能的影响

The Effects of SIRT1 SiRNA on the Bloodfat and Actived Platelets in Insulin Resistance Mice

目的:构建携带小鼠沉默信息调节蛋白1(SIRT1)siRNA的腺病毒载体,并检测其对胰岛素抵抗模型小鼠脂代谢和血小板功能的影响。方法:首先化学合成小鼠SIRT1 shRNA片断,并将目的片断亚克隆入穿梭质粒pShuttle-U6,用PmeⅠ线性化后与pAdeasy-1在BJ5183内同源重组,筛选、鉴定、测序后,在XL10-Gold中扩增重组腺病毒质粒,最后在293细胞内包装扩增为重组腺病毒Ad-SIRT1 SiRNA。用此重组腺病毒感染3T3-L1脂肪细胞,用RT-PCR和ELISA检测其SIRT1 mRNA和蛋白表达从而鉴定其有效性。然后再将重组病毒导入胰岛素抵抗模型小鼠,检测其对小鼠脂代谢和血小板功能的影响。结果:HF组血清胆固醇、甘油三酯、游离脂肪酸和低密度脂蛋白胆固醇显著高于正常对照组(P<0.05),而在HR组上述指标则显著低于HF组(P<0.05)。且HF组高密度脂蛋白胆固醇显著低于正常对照组(P<0.05),在HR组,高密度脂蛋白胆固醇则显著高于HF组(P<0.05)。血小板活化测定结果显示,与正常对照组相比,HF组血小板早期活化程度显著增强(P<0.05),而HR组血小板早期活化程度则比HF组显著减弱(P<0.05),三个组中血小板晚期活化程度无显著性差异(P>0.05)。结论:携带目的基因的腺病毒载体感染脂肪细胞后,能显著影响其SIRT1 mRNA和蛋白表达,并影响胰岛素抵抗模型小鼠脂代谢和血小板功能。

Objective： To construct the RNA interference adenovirus expression vector specific for SIRT1 gene and to observe its effect on the bloodfat and actived platelets in mice.Methods： Firstly,mice SIRT1 gene shRNA fragment were designed,synthesized,and cloned into the vector pshuttle-U6.The adenovirus vector plasmids,Ad-SIRT1 was constructed according to a two-step transforma-tion protocol.Then transfected them into 293 packaging cells to grow adenovirus,which were further multiplied.Then 3T3-L1 cells were infected with the two recombinant adenoviruses respectively.The mRNA expression and protein levels of SIRT1 in these cells were eval-uated by semi-quantitative RT-PCR and ELISA.Further,High insulin-normal glucose clamp was carried out 16 weeks later.Got the blood from jugular vein,analyze the bloodfat and the actived platelets.Results： The recombinant adenoviral plasmids Ad-SIRT1 was suc-cessfully constructed.They remarkably downregulated the expression of SIRT1 at both the mRNA and protein levels in transfected 3T3-L1 cells,and The TG,TC,FFA and LDL-C in HF group is significantly higher than NC group,but it is significantly lower in HR group than in HF group.While the HDL-C in HF group is significantly higher than NC group and HR group.Contrast with the NC group,the earlier actived platelets in HF group is higher but it is no longer higher in HF group.There is no diferenes of the advanced actived platelets in the three groups.Conclusion： The siRNA eukaryotic expression vectors against SIRT1 mRNA effectively inhibited the ex-pression of SIRT1 in 3T3-L1 adipocytes,and down regulated the bloodfat and inhibited the earlier platelets activation in mice.