CAJ | 학술논문

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。
목적：료해결양상태하RAB7단백재은전GFP-RAB7표체재체적신소관상피세포중적표체급위치변화，험증기대세포자서여내탄현상적지시작용。방법：구건GFP-RAB7표체융합단백만병독재체전염신소관상피세포（HK-2），결양처리후，채용RT-qPCR、단백질인적기술화격광공취초기술검측외원기인RAB7재세포내적표체급위치적변화。결과：성공장GFP-RAB7기인적표체재체전염도HK-2세포중，병획득은정표체GFP-RAB7단백적HK-2세포；재결양상태하，RAB7 mRNA적표체경미강저，단백수평몰유변화；격광공취초현미경발현GFP-RAB7단백발생취집，현시자서여내탄적형성。결론：성공건립은정전염적표체GFP-RAB7적HK-2세포계，GFP-RAB7융합단백가지시자서여내탄현상，위연구결양상황하신소관상피세포자서여내탄제공료유효공구。
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.