饥饿诱导人胚肾细胞自噬模型的构建  

Establishment of HEK293 cell line with starvation-induced autophagy

作　　者：黄桢钧 刘慰华[1] 钟赟[1] 刘少军[1] 

机构地区：[1]广州心血管疾病研究所 广州医科大学附属第二医院心内科,广州硕士研究生510260

出　　处：《新医学》2015年第4期221-224,共4页Journal of New Medicine

摘　　要：目的探讨饥饿诱导的人胚肾细胞(HEK293)自噬模型的构建。方法采用Earle's平衡盐溶液(EBSS)分别处理HEK293细胞0、1、2、4、8 h,显微镜下观察细胞形态变化,蛋白免疫印迹法检测自噬微管相关蛋白轻链(LC)3-Ⅱ/LC3-Ⅰ和p62的表达;双荧光mRFP-e GFP-LC3(ptf LC3)质粒转染HEK293细胞24 h后,荧光显微镜观察EBSS饥饿条件下细胞内红色和绿色LC3荧光亮点的变化。结果饥饿处理后,HEK293细胞皱缩、变小变圆,蛋白免疫印迹法可检测到LC3的蛋白表达和LC3-Ⅱ/LC3-Ⅰ比值增加;荧光显微镜可观察到细胞内红色和绿色LC3荧光亮点均增加。结论饥饿可成功诱导HEK293细胞自噬,表现为LC3蛋白表达和LC3-Ⅱ/LC3-Ⅰ比值的增加,以及荧光显微镜下细胞内红色和绿色LC3荧光亮点的增加。Objective To establish HEK293 cell model with starvation-induced autophagy. Methods HEK293 cells were treated with Earle＇s balanced salt solution （EBSS） for 0, 1,2, 4 and 8 h. Then the chan- ges in cell morphology were observed under a microscope. And the expression levels of two autophagy-related proteins including LC3 and p62 were detected by western blotting. After transfeeted with plasmids encoding mRFP-eGFP tandem fluorescent-tagged LC3 （ptfLC3） for 24 h, the changes in LC3 puncta labeled with red and green fluorescence were observed under fluorescence microscopy when HEK293 cells were starved in EB- SS. Results HEK293 cells shrank obviously and became smaller and round after starvation. The expression of LC3 protein and the ratio of LC3-Ⅱ/LC3- I were increased detected by western blotting. The number of LC3 puneta labeled with red and green fluorescence was elevated under fluorescence microscopy. Conclusions Autophagy in HEK293 cells can be successfully induced by starvation, manifested as increased expression of LC3 protein and enhanced ratio of LC3-Ⅱ/LC3-I and elevated number of LC3 pnncta labeled with red and green fluorescence.

关 键 词：饥饿 自噬 自噬微管相关蛋白轻链3 P62 双荧光检测 

分 类 号：R363[医药卫生—病理学]