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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 the ideal cell model of lipometabolism by oil red O and BODIPY staining and to detect the changes of fat in SIRT1 high expression cell line by using high-content instrument. METHODS: Oil red O and BODIPY staining were performed in L-02, HepG2 and Huh7 cells to observe the lipid droplets formation under different concentrations of oleic acid. The constructed SIRT1 overexpression lentiviral vector GV166-SIRT1 and empty vector GV166-infected L-02 cells, qPCR and Western-Blot detection of infected cells SIRT1 expression levels; high intension system detection L-02 SIRT1 and L-02 control cell lines generated lipid droplets fluorescence intensity to determine oleic acid induced The best concentration and the best stimulation time. Results: L-02 cells were found to be more suitable as a cellular model of lipometabolism by Oil Red O staining and BODIPY staining. Successful construction of SIRT1 overexpression lentiviral vector GV166-SIRT1 and empty vector GV166-Control showed that qPCR and Western- The expression level of SIRT1 in the cells was significantly increased after infected with the virus. The fluorescence intensity of lipid droplets in L-02 SIRT1 cells was significantly lower than that of L-02 control when the concentration of oleic acid was 0.4 mM and the induction time was 12 h Differences are maximized. CONCLUSIONS: SIRT1 overexpressing stable transfected cell lines were successfully established and it was demonstrated that SIRT1 overexpression inhibited lipid synthesis.