中文摘要：

【目的】探讨慢病毒介导的shRNA干扰系统抑制脱乙酰基酶1（Sirtuin type 1,Sirt1）表达对猪前体脂肪细胞凋亡的影响。【方法】构建pLentiH1-Sirt1shRNA慢病毒载体,将其感染猪前体脂肪细胞,运用Hoechst33258染色,于荧光显微镜下观察细胞的形态学变化;流式细胞术检测细胞凋亡率;Western印迹检测细胞凋亡标志物Cleaved caspase-3的表达情况。【结果】pLentiH1-Sirt1shRNA慢病毒载体可抑制猪前体脂肪细胞中Sirt1的表达,显微镜下可见细胞体积缩小、染色质凝集、核固缩等典型的凋亡特征;流式细胞术显示细胞凋亡率较空载体对照组明显增加,早期和晚期凋亡细胞百分率分别从4%和9%（P〈0.05）上升至30%和32%（P〈0.05）;Western印迹检测显示Cleaved caspase-3蛋白表达水平显著上调。【结论】慢病毒介导的shRNA可有效地抑制猪前体脂肪细胞中Sirt1的表达,显著促进细胞凋亡,提示Sirt1可能在前体脂肪细胞凋亡中发挥着重要作用。

英文摘要：

[Objective] The study was to investigate the effects of Sirt1 inhibition by lentivirus-mediated shRNA vector on apoptosis of porcine preadipocytes.[Method] pLentiH1-Sirt1 shRNA lentivirus vector was constructed to infect porcine preadipocytes.The morphological changes of cells were observed under fluorescence microscope after Hoechst 33258 staining;flow cytometer was used to assess the percentages of apoptotic cells;expression of apoptotic marker Cleaved caspase-3 in porcine preadipocytes was examined by Western blotting analysis.[Result] After Sirt1 expression was inhibited by lentiviral Sirt1 shRNA infection.Preadipocytes showed characteristic morphological features of apoptosis under fluorescence microscope,such as reduced cell volume,chromatin condensation and nuclear shrinkage.Apoptosis rate in cells infected with Sirt1 shRNA was significantly increased compared with that of empty vector group,and the early apoptotic and late apoptotic cell fractions increased from 4% and 9%（P〈0.05） to 30% and 32%（P〈0.05）,respectively,assessed with flow cytometric analysis.Western blotting analysis showed that inhibition of Sirt1 also upregulated the Cleaved caspase-3 expression.[Conclusion] Lentivirus-mediated shRNA vector effectively inhibited Sirt1 expression and remarkably induced apopotosis in porcine preadipocytes.These results suggest that Sirt1 may play an important role in the apoptosis of preadipocytes.