稳定表达小鼠PNPLA7的Huh7细胞系的构建

目的:构建稳定表达小鼠PNPLA7-Flag蛋白的人类肝癌细胞Huh7细胞系,观察小鼠PNPLA7在Huh7细胞中的过表达以及对Huh7细胞脂质含量的影响?方法:构建pCMV6 -PNPLA7-Flag真核表达载体,应用脂质体转染技术将该质粒导入Huh7细胞,随后通过G418筛选稳定表达的细胞系,运用Western blot方法检测PNPLA7蛋白表达;并通过油酸处理及油红染色观察过表达PNPLA7蛋白对Huh7细胞中脂质含量的影响?结果:成功获得2株稳定过表达PNPLA7-Flag的Huh7细胞系;Huh7细胞中过表达PNPLA7蛋白,在油酸处理后细胞内脂滴含量减少?结论:稳定表达小鼠PNPLA7的Huh7细胞系构建成功;小鼠PNPLA7在Huh7细胞中的表达会引起细胞中脂质含量减少,从而为后续研究PNPLA7蛋白在脂代谢通路的功能及机制提供可靠的细胞模型?

Establishment of Huh7 cell line stably expressing murine PNPLA7 protein

Objective:To establish a stable PNPLA7-expressed human hepatoma cell line (Huh7) and observe the effect of stable overexpression of PNPLA7 on the lipid contents in the cell line. Methods: pCMV6-PNPLA7-Flag plasmid was constructed and transfected into Huh7 cells with lipofectamine 2000 reagent. After G418 selection, mRNA and protein expression levels of PNPLA7 were examined by Western blot in these stable cell lines. Treated cells with oleic acid and the lipid contents in these cell lines were observed by oil red O staining. Results: Two lines stably expressing PNPLA7 were gained,and showed high expression level of PNPLA7 by Western blot analysis. The decreased lipid contents were observed in these cell lines after oleic acid treatment by oil red O staining. Conclusion: Huh7 cell lines stably expressing PNPLA7- Flag were established successfully. Stable overexpression of PNPLA7 may lead to the decreased lipid content in Huh7 cells after oleic acid treatment. It can be a reliable cell model for the study of functions and mechanisms of PNPLA7 in lipid metabolic pathways.