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目的:研究ATP缺失时大鼠近端肾小管上皮细胞(NRK52E)肌动蛋白磷酸化水平的改变。方法:建立细胞的体外ATP缺失模型,用免疫印迹技术检测细胞内骨架组分及胞浆组分中肌动蛋白的分布变化;用二维电泳法分离并比较细胞内磷酸化和非磷酸化的肌动蛋白含量的改变;用免疫共沉淀法及免疫印迹技术检测ATP缺失后细胞内肌动蛋白酪氨酸、苏氨酸及丝氨酸磷酸化水平变化。结果:ATP缺失处理后细胞内F-肌动蛋白含量逐渐增加,G-肌动蛋白含量逐渐减少;ATP缺失细胞内磷酸化肌动蛋白量明显增加,肌动蛋白的磷酸化程度随ATP缺失时间延长而增加;ATP缺失处理后肌动蛋白酪氨酸磷酸化水平逐渐升高,且与ATP缺失程度呈相一致;肌动蛋白苏氨酸磷酸化水平无明显变化。结论:ATP缺失后近端肾小管上皮细胞肌动蛋白出现聚合过程,可能与细胞中肌动蛋白磷酸化酪氨酸水平增加有关。
AIM: To investigate the changes of actin phosphorylation in rat proximal renal tubular epithelial cells (NRK52E) induced by ATP deficiency. METHODS: The in vitro ATP-deficient model of cells was established. The distribution of actin in cytoskeleton and cytoplasm was detected by Western blotting. The two-dimensional electrophoresis was used to separate and compare intracellular phosphorylated and non-phosphorylated The changes of actin content were detected by immunoprecipitation and Western blotting. The levels of actin tyrosine, threonine and serine phosphorylation were detected by immunoprecipitation and immunoblotting. Results: The content of F-actin in the cells increased gradually and the content of G-actin decreased gradually after the ATP-deficient treatment. The amount of phosphorylated actin in ATP-deficient cells increased obviously. The phosphorylation level of actin decreased with the ATP-deficient time Prolonged and increased; the level of actin tyrosine phosphorylation gradually increased after ATP-deficient treatment, and it was consistent with the degree of ATP deficiency; and actin-threonine phosphorylation level did not change significantly. CONCLUSION: The process of actin polymerization in proximal tubular epithelial cells after ATP depletion may be related to the increase of actin phosphorylation tyrosine level in cells.