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

doi:10.13481/j.1671-587x.20150619

吉林大学学报(医学版) ›› 2015, Vol. 41 ›› Issue (06): 1195-1200.doi: 10.13481/j.1671-587x.20150619

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

黄志立¹, 张丽君¹, 伍丽华², 王妍¹, 杨汝德²

1. 深圳职业技术学院应用化学与生物技术学院, 广东深圳 518055;
2. 华南理工大学生物科学与工程学院, 广东广州 510640

收稿日期:2015-04-21 发布日期:2016-01-11
通讯作者: 张丽君,副教授(Tel:0755-26019560,E-mail:c7zlj@szpt.edu.cn) E-mail:c7zlj@szpt.edu.cn
作者简介:黄志立(1974-),女,四川省兴文县人,副教授,工学博士,主要从事生物技术应用方面的研究。
基金资助:
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)

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

HUANG Zhili¹, ZHANG Lijun¹, WU Lihua², WANG Yan¹, YANG Rude²

1. School of Applied Chemistry and Biological Technology, Shenzhen Polytechnic, Shenzhen 518055, China;
2. School of Bioscience and Bioengineering, South China University of Sciences and Technology, Guangzhou 510640, China

Received:2015-04-21 Published:2016-01-11

摘要/Abstract

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。

关键词: 血管紧张素原, RNA干扰, 3T3-L1, 分化

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 U6 promoter-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 was 43.86% 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.

Key words: angiotensinogen, RNA interference, 3T3-L1, differentiation

中图分类号:

黄志立, 张丽君, 伍丽华, 王妍, 杨汝德. 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[J]. 吉林大学学报(医学版), 2015, 41(06): 1195-1200.

HUANG Zhili, ZHANG Lijun, WU Lihua, WANG Yan, YANG Rude. Influence of angiotensinogen gene silencing on differentiation of preadipocytes 3T3-L1[J]. Journal of Jilin University Medicine Edition, 2015, 41(06): 1195-1200.

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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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