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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 adenoviral vector carrying mouse SIRT1 siRNA and to detect its effect on lipid metabolism and platelet function in mice with insulin resistance. Methods: The SIRT1 shRNA fragment was chemically synthesized and subcloned into the shuttle plasmid pShuttle-U6. After homologous recombination with pAdeasy-1 in BJ5183 by PmeⅠ, the recombinant plasmid was screened, identified and sequenced. Gold and amplify the recombinant adenovirus plasmid Ad-SIRT1 SiRNA in 293 cells. The recombinant adenovirus was used to infect 3T3-L1 adipocytes. The expression of SIRT1 mRNA and protein was detected by RT-PCR and ELISA. Then, the recombinant virus was introduced into insulin resistance model mice to test their effects on lipid metabolism and platelet function in mice. Results: The levels of serum cholesterol, triglyceride, free fatty acid and low density lipoprotein cholesterol in HF group were significantly higher than those in normal control group (P <0.05), while those in HF group were significantly lower than those in HF group (P <0.05). HF group had significantly lower HDL cholesterol than HF group (P <0.05). HF group had significantly higher HDL cholesterol than HF group (P <0.05). The results of platelet activation showed that the early activation of platelet in HF group was significantly higher than that in normal control group (P <0.05), while the activation of platelet in HF group was significantly weaker than that in HF group (P <0.05) There was no significant difference in late platelet activation (P> 0.05). CONCLUSION: The adenovirus vector carrying the target gene can significantly affect the expression of SIRT1 mRNA and protein, as well as affect lipid metabolism and platelet function in mice with insulin resistance.