Fsp27基因沉默载体的构建及其对细胞脂解的影响研究

doi:10.13560/j.cnki.biotech.bull.1985.2019-0369

生物技术通报 ›› 2020, Vol. 36 ›› Issue (1): 88-94.doi: 10.13560/j.cnki.biotech.bull.1985.2019-0369

Fsp27基因沉默载体的构建及其对细胞脂解的影响研究

许祥¹, 董维鹏¹, 张少华¹, 冯晨毅¹, 刘田福², 燕炯¹

1. 山西医科大学公共卫生学院,太原 030001;
2. 山西医科大学动物实验中心,太原 030001

收稿日期:2019-04-28 出版日期:2020-01-26 发布日期:2020-01-08
作者简介:许祥,硕士研究生,研究方向：肥胖相关疾病分子学;E-mail：1079269247@qq.com
基金资助:
山西省国际科技合作项目（2014081050-1）,山西医科大学科技创新基金项目（01201504）

Construction of Fsp27 Gene Silencing Vector and Its Effect on Cell Lipolysis

XU Xiang¹, DONG Wei-peng¹, ZHANG Shao-hua¹, FENG Chen-yi¹, LIU Tian-fu², YAN Jiong¹

1. School of Public Health,Shanxi Medical University,Taiyuan 030001;
2. Laboratory Animal Center of Shanxi Medical University,Shanxi Medical University,Taiyuan 030001

Received:2019-04-28 Published:2020-01-26 Online:2020-01-08

摘要/Abstract

摘要： 构建脂肪特异性蛋白27（Fat-specific protein of 27,Fsp27）基因沉默载体,研究沉默Fsp27基因表达对3T3-L1细胞脂解的影响,并对其作用机制进行探究。采用RNAi技术,构建Fsp27基因真核干扰载体,下调Fsp27基因的表达。“鸡尾酒”法诱导3T3-L1前脂肪细胞分化为成熟脂肪细胞。脂质体转染脂肪细胞,油红O染色脂滴,酶法测定细胞中甘油及甘油三酯的含量。Western blot法检测细胞中Fsp27、HSL、ATGL和PPARγ的蛋白表达。Western blot结果显示：阳性sh-Fsp27干扰载体均能有效下调Fsp27的表达,且伴随细胞内ATGL和PPARγ的表达量升高（P<0.05）,其中sh-Fsp27-2的沉默效果最好;酶学方法检测结果显示：阳性sh-Fsp27干扰组细胞中甘油三酯含量下降,甘油含量升高（P<0.05）;油红O染色结果发现：空白对照组与阴性对照组均有大脂滴堆积,阳性sh-Fsp27组小脂滴分布广泛,未见明显的大脂滴。sh-Fsp27-2组基因沉默载体的沉默效果最好,Fsp27基因沉默可以加快3T3-L1细胞的脂解速率,其主要是通过抑制脂滴融合和增强ATGL酶的水解来完成对脂解的调控。

关键词: 脂肪特异性蛋白27, RNA干扰, 3T3-L1前脂肪细胞, 脂解

Abstract: This work is aimed to construct the fat-specific protein of 27（Fsp27）gene silencing vector and to investigate the effect of Fsp27 gene expression on the lipolysis of 3T3-L1 cells as well as explore its mechanism. RNAi technology was used to construct the eukaryotic interference vector of Fsp27 gene to down-regulate the expression of the Fsp27 gene. The “cocktail” method induced differentiation of 3T3-L1 preadipocytes into mature adipocytes. Liposomes were transfected into adipocytes,oil red O stained lipid droplets,and enzymatic method was employed to determine the content of glycerol and triglyceride in cells. Western blot analysis was used to detect the protein expression of Fsp27,HSL,ATGL and PPARγ in cells. The results from Western blot showed that the positive sh-Fsp27 vector down-regulated the expression of Fsp27,while the expression of ATGL and PPARγ increased（P<0.05）,and the silencing results by sh-Fsp27-2 was the best. The results of enzymology demonstrated that the content of triglyceride in the positive sh-Fsp27 interference group decreased,and the content of glycerol increased（P<0.05）. The results of oil red O staining showed that there were large lipid droplets accumulated in the blank control group and the negative control group,while the distribution of small lipid droplets in the positive sh-Fsp27 group was major,and no obvious big fat drops was observed. In conclusion,the silencing effect by sh-Fsp27-2 is the best,Fsp27 gene silencing may accelerate the lipolysis rate of 3T3-L1 cells,which is mainly achieved by inhibiting the fusion among small lipid droplets and increasing the ATGL-mediated hydrolysis .

Key words: Fsp27, RNAi, 3T3-L1 preadipocytes, lipolysis

许祥, 董维鹏, 张少华, 冯晨毅, 刘田福, 燕炯. Fsp27基因沉默载体的构建及其对细胞脂解的影响研究[J]. 生物技术通报, 2020, 36(1): 88-94.

XU Xiang, DONG Wei-peng, ZHANG Shao-hua, FENG Chen-yi, LIU Tian-fu, YAN Jiong. Construction of Fsp27 Gene Silencing Vector and Its Effect on Cell Lipolysis[J]. Biotechnology Bulletin, 2020, 36(1): 88-94.

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Fsp27基因沉默载体的构建及其对细胞脂解的影响研究

Construction of Fsp27 Gene Silencing Vector and Its Effect on Cell Lipolysis

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