稳转GFP-RAB7的肾小管上皮细胞在缺氧状态下RAB7的表达及位置变化-东南大学学报医学版

稳转GFP-RAB7的肾小管上皮细胞在缺氧状态下RAB7的表达及位置变化

单位：东南大学医学院, 江苏南京 210009

分类号：R361.3;R364.4

出版年·卷·期（页码）：2015·34·第五期（710-713）

摘要：

目的:了解缺氧状态下RAB7蛋白在稳转GFP-RAB7表达载体的肾小管上皮细胞中的表达及位置变化,验证其对细胞自噬与内吞现象的指示作用。方法:构建GFP-RAB7表达融合蛋白慢病毒载体转染肾小管上皮细胞(HK-2),缺氧处理后,采用RT-qPCR、蛋白质印迹技术和激光共聚焦技术检测外源基因RAB7在细胞内的表达及位置的变化。结果:成功将GFP-RAB7基因的表达载体转染到HK-2细胞中,并获得稳定表达GFP-RAB7蛋白的HK-2细胞;在缺氧状态下,RAB7 mRNA的表达轻微降低,蛋白水平没有变化;激光共聚焦显微镜发现GFP-RAB7蛋白发生聚集,显示自噬与内吞的形成。结论:成功建立稳定转染的表达GFP-RAB7的HK-2细胞系,GFP-RAB7融合蛋白可指示自噬与内吞现象,为研究缺氧状况下肾小管上皮细胞自噬与内吞提供了有效工具。

Objective: To establish the HK-2 cell line (human proximal tubular cells) stably transfected an expressing vector of human GFP-RAB7 fused protein,and detect its indicative function of autophagy and endocytosis under hypoxia. Methods: Lentiviral expression vector of human GFP-RAB7 fused protein was constructed, and then stably transfected to HK-2 cell line. The expression and location of GFP-RAB7 were detected by RT-qPCR, Western blot and laser scanning confocal microscope(LSCM). Results: The HK-2 cells transfected the vector stably expressed GFP-RAB7 fused protein. These results indicated that the level of RAB7 mRNA was decreased under hypoxic conditions, while hypoxia did not affect the expression of total amount of RAB7 protein. The result of LSCM showed the aggregation of GFP-RAB7 and formation of autophagosome in transfected HK-2 cells under hypoxia. Conclusion: We have successfully constructed a stable transfected HK-2 cell line which expresses GFP-RAB7 protein. The fuse protein could be a good indicator for autophagy and endocytosis. It may provide a simple and effective method for studying autophagy and endocytosis in cells.

参考文献：

[1] FINE L G,BANDYOPADHAY D,NORMAN J T.Is there a common mechanism for the progression of different types of renal diseases other than proteinuria?Towards the unifying theme of chronic hypoxia[J].Kidney Int,2000,75:S22-26.
[2] TANAKA S,TANAKA T,NANGAKU M.Hypoxia as a key player in the AKI-to-CKD transition[J].Am J Physiol Renal Physiol,2014,307(11):F1187-1195.
[3] LI J,FAN R,ZHAO S,et al.Reactive oxygen species released from hypoxic hepatocytes regulates MMP-2 expression in hepatic stellate cells[J].Int J Mol Sci,2011,12:2434-2447.
[4] MCEWAN D G,DIKIC I.PLEKHM1:adapting to life at the lysosome[J].Autophagy,2015,11(4):720-722.
[5] HYTTINEN J M,NIITTYKOSKI M,SALMINEN A,et al.Maturation of autophagosomes and endosomes:a key role for Rab7[J].Biochim Biophys Acta,2013,1833(3):503-510.
[6] MIZUSHIMA N,YOSHIMORI T,LEVINE B.Methods in mammalian autophagy research[J].Cell,2010,140(3):313-326.
[7] CAMPANHOLLE G,LIGRESTI G,GHARIB S A.et al.Mechanisms of Tissue Fibrosis.3.Novel mechanisms of kidney fibrosis[J].Am J Physiol Cell Physiol,2013,304(7):C591-C603.
[8] MIZUSHIMA N.Methods for monitoring autophagy[J].Int J Biochem Cell Biol,2004,36(12):2491-2502.
[9] 汪昌丽,满娜,温龙平.纳米材料诱导细胞自噬的效应及生物学功能[J].东南大学学报:医学版,2011,30(1):200-208.

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