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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p CMV6-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。
OBJECTIVE: To construct Huh7 human hepatoma cell line stably expressing mouse PNPLA7-Flag protein and to observe the overexpression of PNPLA7 in Huh7 cells and the effect on the lipid content of Huh7 cells. Methods: The eukaryotic expression vector p CMV6-PNPLA7-Flag was constructed. The plasmid was transfected into Huh7 cells by lipofection technique. Stably expressing cell lines were screened by G418. The expression of PNPLA7 protein was detected by Western blot. Effects of overexpression of PNPLA7 protein on lipid content in Huh7 cells were observed by acid treatment and oil red staining. RESULTS: Two Huh7 cell lines stably overexpressing PNPLA7-Flag were successfully obtained. PNPLA7 protein was overexpressed in Huh7 cells, and the amount of lipid droplets in the cells was decreased after oleic acid treatment. CONCLUSION: The Huh7 cell line stably expressing mouse PNPLA7 was successfully constructed. The expression of PNPLA7 in Huh7 cells caused a decrease of the lipid content in the cells, which provided a reliable cell for further studies on the function and mechanism of PNPLA7 protein in lipid metabolism pathway model.