论文部分内容阅读
目的研究肾小管上皮细胞ATP耗竭再恢复时热休克蛋白(HSP)72与桩蛋白(Paxillin)的相互作用和意义。方法应用代谢抑制剂暂时性阻断肾小管上皮细胞ATP的生成,引起细胞内的ATP耗竭;换用含10 mmol／L葡萄糖的DMEM培养液,使细胞内ATP再恢复;以热处理细胞或编码HSP72 RNA的腺病毒感染细胞,诱导细胞高表达HSP72。Western印迹检测HSP72水平;间接免疫荧光和Western印迹检测Paxillin在细胞内的分布变化。免疫共沉淀观察HSP72与Paxillin的相互作用。结果肾小管上皮细胞ATP耗竭再恢复时,对照组细胞内的Paxillin由局部黏附结构区域向细胞浆内弥散分布;HSP72由细胞浆转移至细胞膜。细胞高表达HSP72后,HSP72在细胞浆和细胞膜中的蛋白含量均增加(P<0．05);Paxillin由细胞浆向细胞膜的转移明显减少(P<0．05);Paxillin在细胞内的正常分布改善;HSP72和Paxillin之间的相互作用显著增加(P<0．05)。结论肾小管上皮细胞ATP耗竭再恢复时,HSP72可保持Paxillin在细胞内的正常分布特点和区域,其机制可能是HSP72增加与Paxillin的相互作用,发挥分子伴侣的功能。
Objective To investigate the interaction and significance of heat shock protein (HSP) 72 and Paxillin in renal tubular epithelial cells after ATP depletion and recovery. Methods Metabolic inhibitors temporarily blocked the production of ATP in renal tubular epithelial cells and caused intracellular ATP depletion. The medium was resuspended with DMEM supplemented with 10 mmol / L glucose, and the cells were treated with heat or HSP72 RNA-infected adenovirus-induced cells induce HSP72 overexpression. The level of HSP72 was detected by Western blotting. The distribution of Paxillin in cells was detected by indirect immunofluorescence and Western blot. Coimmunoprecipitation observed HSP72 and Paxillin interaction. Results When the renal tubular epithelial cells were depleted of ATP and then resumed, the Paxillin in the control group was diffused into the cytoplasm by the region of local adhesive structure. HSP72 was transferred from the cytoplasm to the cell membrane. After the cells were highly expressed HSP72, the protein content of HSP72 in the cytoplasm and cell membrane were increased (P <0.05); the migration of Paxillin from the cytoplasm to the cell membrane was significantly reduced (P <0.05); Paxillin was normal in the cell Distribution improved; the interaction between HSP72 and Paxillin increased significantly (P <0.05). Conclusion HSP72 can maintain the normal distribution and region of Paxillin in the renal tubular epithelial cells. The mechanism may be that HSP72 increases the interaction with Paxillin and plays a role of chaperone.