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目的:在肝脏L-02 SIRT6-KO细胞系中,观察SIRT6在肝脏脂滴形成中的作用,初步研究在肝脏脂质代谢中SIRT6相关的转录差异基因发挥作用的分子机制。方法:利用基因敲除技术TALEN技术,构建肝脏L-02 SIRT6特异性敲除细胞系,并经q PCR和Western blot检测其表达水平;利用油酸刺激L-02 SIRT6-KO细胞及其对照组,体外模拟肝脏脂肪变的条件,并经高内涵和油红染色验证其脂滴形成能力;利用基因芯片检测L-02 SIRT6-KO及其对照细胞系中相关差异基因,并经生物信息学分析筛选脂代谢相关差异基因。结果:建立人肝脏SIRT6特异性敲除细胞系,q PCR显示m RNA下降50%,但Western blot证明SIRT6完全敲除;BODIPY实验及油红O染色发现,L-02 SIRT6-KO细胞比对照组其脂滴形成数量增多,高内涵荧光检测显示L-02 SIRT6-KO细胞中脂滴荧光强度比对照组增强(176.38±2.55 vs 104.26±2.08);通过对差异转录基因分析,发现了一组在脂质代谢中和SIRT6相关的关键基因如STEAP4、HMGA2、PDE1A、INSL4等。结论:成功建立人肝脏L-02 SIRT6-KO细胞系,并经实验证明SIRT6缺失能够促进脂滴形成;筛选到一组和SIRT6相关参与脂质代谢的关键基因。
OBJECTIVE: To observe the role of SIRT6 in the formation of lipid droplets in liver L-02 SIRT6-KO cell line and to study the molecular mechanism of SIRT6-related transcriptional differences in liver lipid metabolism. Methods: Liver L-02 SIRT6-specific knockout cell line was constructed by gene knockout technique TALEN. The expression of L-02 SIRT6-KO cells was detected by qPCR and Western blot. , The condition of hepatic steatosis was simulated in vitro, and its lipid droplet formation ability was verified by high content and oil red staining. The related differential genes in L-02 SIRT6-KO and its control cell line were detected by gene chip and analyzed by bioinformatics Screening for lipid metabolism related genes. Results: The human liver SIRT6-specific knockout cell line was established. Q-PCR showed that m RNA decreased by 50%, but Western blot showed that SIRT6 knocked out completely. BODIPY assay and oil red O staining showed that L-02 SIRT6- The number of lipid droplets increased, and the high content of fluorescence detection showed that the fluorescence intensity of lipid droplets in L-02 SIRT6-KO cells was stronger than that of the control group (176.38 ± 2.55 vs 104.26 ± 2.08) .According to the differential transcriptional gene analysis, Lipid metabolism and SIRT6-related key genes such as STEAP4, HMGA2, PDE1A, INSL4 and so on. Conclusion: The human liver L-02 SIRT6-KO cell line was successfully established and the SIRT6 deletion was found to promote lipid droplet formation. A group of key genes related to SIRT6 involved in lipid metabolism were screened.