【摘要】 目的:建立稳定表达GFP-LC3的人永生化角质形成细胞HaCaT细胞系。方法:将构建的pcDNA3.1-GFP-LC3真核表达载体转入HaCaT细胞,经G418筛选稳定表达的细胞系。HaCaT细胞中GFP-LC3的表达分别用荧光显微镜与Western blot方法检测,并利用该稳定表达的细胞系观察验证Rapamycin对细胞发生自噬透射电镜超微结构的变化。结果:获得了3株转染并经G418反复筛选的HaCaT细胞系,在倒置荧光显微镜下观察可见绿色荧光细胞的表达率在95%以上,Western blot结果证实了GFP-LC3融合蛋白的表达。Western blot和激光共聚焦显微镜均证明Rapamycin可以诱导自噬的发生。透射电镜细胞超微结构的观察表明Rapamycin可以有效地诱导HaCaT-LC3细胞自噬的发生。结论:成功构建GFP-LC3稳定表达的HaCaT系,该细胞系可以作为研究人角质形成细胞自噬功能的一种细胞模型。更多还原

【Abstract】 Autophagy has been reported to contribute to wound repair and regeneration,but in vivo,it is infeasible to detect autophagy of human patient’s skin.HaCaT cell line is spontaneously immortalized human keratinocytes from human skin.To establish a stable GFP-LC3-expressed HaCaT cell line.The pcDNA3.1-GFP-LC3 plasmid was constructed and transfected into HaCaT cell with transfection reagent.The stable transfectants were screened by G418.The GFP-LC3 protein expression was analyzed by Western blot.The fluorescent signals were detected by inverted fluorescence microscope.Rapamycin-induced autophagy was detected by confocal microscope,Western blot and transmission electronic microscope(TEM).Selected by G418,3 transfected cell lines showed high expression level of GFP-LC3,as demonstrated by Western blot analysis.More than 95% cells showed positive fluorescent signals under inverted fluorescence microscope.The formation of autophagosomes and the increases in the conversion of LC3-I to LC3-II was observed in the constructed cells when treated with Rapamycin.Rapamycin-induced autophagy in HaCaT-LC3 cell line was detected by TEM.A HaCaT cell line stably expressing GFP-LC3(named HaCaT-LC3)was constructed successfully.The HaCaT-LC3 cell line may act as a cellular model to study the autophagy in epidermic cell exactly.更多还原