| 马静波 霍静 张松 丁美玲 聂勇战.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[J].,2014,14(9):1616-1621 |
| 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 OverexpressionLentiviral and Its Effect on Fat Synthesis |
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| DOI： |
| 中文关键词: 油红O BODIPY SIRT1 慢病毒 高内涵监测系统 |
| 英文关键词: Oil red O BODIPY SIRT1 Lentiviral High Content Analysis |
| 基金项目:科技部重大国际合作项目（81110200）：肝脏脂质代谢障碍中SIRT1 相关分子的组学研究 |
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| 中文摘要: |
| 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。 |
| 英文摘要: |
| 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 SIRT1 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-SIRT1 and GV166-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-sirt1 group was significantly lower than that in L-02-control group, while the
difference in fluorescence intensity between the L-02-sirt1 L-02-control was largest.Conclusion:The SIRT1 overexpressing stably
transfected cell lines were successful established and overexpression SIRT1 can inhibit fat synthesis. |
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