【摘要】 目的:构建靶向血管紧张素原(AGT)基因的RNA干扰质粒,研究AGT基因对鼠前脂肪3T3-L1分化的影响。方法:设计靶向AGT的小分子RNA干扰片段(AGT-shRNA),以未靶向任何DNA的小分子RNA(Non-shRNA)为对照,利用载体psiRNA-U6.1/Neo构建重组表达质粒,转染鼠前脂肪细胞3T3-L1并筛选到稳定转染细胞株,RT-PCR和SDS-PAGE法分别检测AGT基因沉默效果,通过显微镜观察和脂肪细胞分化标志物检测来确定AGT基因干扰对3T3-L1细胞分化的影响。结果:成功构建AGT干扰质粒,与对照组比较,AGT基因转录水平受到显著抑制(P<0.05),AGT蛋白表达水平也仅为对照的43.86%。AGT基因干扰后3T3-L1细胞脂质水平和甘油-3-磷酸脱氢酶(GPDH)活性仍随分化时间延长呈上升趋势,但增长量明显低于对照组(P<0.05)。结论:RNA干扰AGT基因可明显抑制3T3-L1前脂肪细胞的分化,提示脂肪组织中的肾素-血管紧张素系统(RAS)与脂肪代谢有重要关联。更多还原

【Abstract】 Objective To construct the angiotensinogen(AGT)-targeting RNA interfering plasmids,and to explore the effect of AGT gene on the differentiation of preadipocytes 3T3-L1 in the mice.Methods The small hairpin RNA(AGT-shRNA)interference plasmids,with a scrambled Non-shRNA as control,were constructed on the basis of the activity of U6promoter-driven expression vector psiRNA-U6.1/Neo and transfected into the mouse preadipocytes 3T3-L1.Two cell lines were generated from stable transfections.RT-PCR and SDS-PAGE were performed to monitor the mRNA and protein expressions of AGT gene,respectively.The effect of adipose AGT on the differentiation of 3T3-L1 was investigated through microscopic observation and profiling of adipocyte differentiation markers.Results Both AGT-shRNA and Non-shRNA interference vectors were successfully constructed;the expression of AGT mRNA was inhibited(P<0.05)and the expression of AGT protein was43.86% of control.Both adipocyte differentiation markers,intracytoplasmic lipid levels and glycerol-3-phophate dehydrogenase(GPDH)activities were increased during the differentiation of preadipocytes 3T3-L1,but the data were lower than those in control group(P<0.05).Conclusion AGT gene silencing can significantly inhibit the differentiation of preadipocytes 3T3-L1,which demonstrates that there is important relationship between adipocyte metabolism and renin-angiotensin system(RAS)in adipose tissue.更多还原