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RNA干扰血管紧张素原基因表达对前脂肪细胞3T3-L1分化的影响

Influence of angiotensinogen gene silencing on differentiation of preadipocytes 3T3-L1

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【作者】 黄志立张丽君伍丽华王妍杨汝德

【Author】 HUANG Zhili;ZHANG Lijun;WU Lihua;WANG Yan;YANG Rude;School of Applied Chemistry and Biological Technology,Shenzhen Polytechnic;School of Bioscience and Bioengineering,South China University of Sciences and Technology;

【机构】 深圳职业技术学院应用化学与生物技术学院华南理工大学生物科学与工程学院

【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。

【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.

【关键词】 血管紧张素原RNA干扰3T3-L1分化
【Key words】 angiotensinogenRNA interference3T3-L1differentiation
【基金】 广东省教育厅产学研结合项目资助课题(2012B091100408);深圳市科技计划项目资助课题(07KJba160);广东省深圳市创新委新兴产业公共技术服务平台项目资助课题(GGJS20130331152344401)
  • 【文献出处】 吉林大学学报(医学版) ,Journal of Jilin University(Medicine Edition) , 编辑部邮箱 ,2015年06期
  • 【分类号】R33
  • 【被引频次】1
  • 【下载频次】73
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