SIRT1过表达慢病毒稳转细胞系的构建及其对脂肪合成的影响

Construction of Stable Transfected Cell Line of SIRT1 Overexpression Lentiviral and Its Effect on Fat Synthesis

目的:通过油红O及BODIPY染色选取脂肪代谢理想的细胞模型,并运用高内涵仪器检测SIRT1高表达细胞系中脂肪的变化。方法:在L-02、HepG2、Huh7细胞中进行油红O和BODIPY染色,观察在不同油酸浓度下脂滴的生成情况;将构建成功的SIRT1过表达慢病毒载体GV166-SIRT1及空载体病毒GV166-Control感染L-02细胞,qPCR及Western-Blot检测感染细胞中SIRT1的表达水平;高内涵系统检测L-02 SIRT1和L-02 control细胞系产生的脂滴荧光强度,以确定油酸诱导的最佳浓度及最佳刺激时间。结果:通过油红O及BODIPY染色发现L-02细胞更适宜作为脂肪代谢的细胞模型;成功构建SIRT1过表达慢病毒载体GV166-SIRT1及空载体病毒GV166-Control,qPCR及Western-Blot检测显示转染病毒后SIRT1在细胞中的表达水平明显升高;在油酸刺激浓度0.4mM、诱导时间12h时,L-02 SIRT1细胞中脂滴的荧光强度明显较L-02 control为低,且这种差异达到最大化。结论:成功建立SIRT1过表达稳定转染细胞系,并证明高表达SIRT1能够抑制脂肪合成。

Objective： To select a suitable cell model of fatty metabolism by oil red O staining and Bodipy staining, and detect the changes of adipose in S1RT1 overexpression cell lines by High Content Analysis. Methods： The lipid droplets generation of HepG2, Huh7 and L-02 cell lines was observed by using oil red O staining and Bodipy staining under different oleic acid stimulation concentrations; SIRT1 overexpression lentiviral vector GV166-SIRT1 and control lentiviral vector GV166-Control was constructed to infect L-02 cell line, and the expression levels of SIRT1 within infected the cells were detected by qPCR and Western-Blot ; High Content Analysis was performed to detect the fluorescence intensity of lipid droplets within L-02 SIRT1 and L-02 control cell lines in order to determine the optimal concentration of oleic acid-induced stimulation and the best time. Results： By oil red O and bodipy staining, it was found that L-02 was more suitable for a cell model of liver lipid metabolism. Lentiviral vectors GV166-SIRT 1 and GV 166-Control were confirmed to be successfully constructed and SIRT1 overexpression lentiviral vector was proven to be capable of raising significantly the SIRT1 expression level within the L-02 cells by qPCR and Western-Blot. When the concentration of oleic acid was 0.4mM and the treatment duration was 12h, the fluorescence intensity in L-02-sirtl group was significantly lower than that in L-02-control group, while the difference in fluorescence intensity between the L-02-sirtl L-02-control was largest. Conclusions： The SIRT1 overexpressing stably transfected cell lines were successful established and overexpression SIRT1 can inhibit fat synthesis.