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目的:探讨肾上管上皮细胞(RTEs)S期细胞凋亡过度与G1/S界面细胞低氧直接暴露的关系。方法:采用NaCN体外细胞化学性低氧法进行G1/S新生猪RTEs低氧干预研究,分别应用反相高效液相法、流式细胞术细胞DNA含量PI染色分析法和流式细胞术间接细胞免疫化学法进行低氧/再给氧各观察点细胞内ATP含量、细胞周期时相和凋亡细胞分布率以及热休克蛋白(HSP27和HSP70)表达量的检测。并采用磷蛋白磷酸酶(PP2A)抑制剂斑蝥素进行NaCN介导的细胞蛋白质脱磷酸化抑制研究,比较HSP27和HSP70在磷酸化与脱磷酸化状态下细胞凋亡的发生率。结果:受低氧直接暴露的G1/S新生猪RTEs在S期细胞凋亡比率显著高于PP2A抑制组和空白对照组(P<0·01),此时细胞内ATP含量显著低于后两组(P<0·05),而细胞内HSP27和HSP70的表达量则均高于后两组(P<0·01)。结论:新生猪RTEs发生S期细胞凋亡过度与其G1/S低氧直接暴露密切相关,其可能机制与NaCN化学性低氧介导的S期HSP27和HSP70的脱磷酸化有关。
Aims: To investigate the relationship between over-apoptosis of S-phase epithelial cells in renal epithelial cells (RTEs) and direct exposure to hypoxia at G1 / S interface. Methods: The hypoxic intervention of G1 / S neonatal piglets with RTEs was performed by using cytochemical hypoxia method in vitro. Reverse transcription-phase high performance liquid chromatography (RP-HPLC), flow cytometry (FCM), PI staining and flow cytometry Immunochemistry method was used to detect the content of intracellular ATP, the distribution of cell cycle phase and apoptotic cells and the expression of heat shock proteins (HSP27 and HSP70) in each observation point under hypoxia / reoxygenation. The phosphoprotein phosphatase (PP2A) inhibitor cantharidin was used to study NaCN-mediated dephosphorylation of cellular proteins. The incidence of apoptosis of HSP27 and HSP70 in phosphorylation and dephosphorylation was compared. Results: The apoptosis rate of RTEs in G1 / S neonatal piglets exposed to hypoxia at S phase was significantly higher than that of PP2A inhibition group and blank control group (P <0.01), and the intracellular ATP content was significantly lower than that of the latter two (P <0.05), while the expression of HSP27 and HSP70 in the cells were higher than the latter two groups (P <0.01). CONCLUSIONS: S phase apoptosis in newborn piglets is closely related to its direct exposure to G1 / S hypoxia. The possible mechanism is related to the dephosphorylation of HSP27 and HSP70 mediated by NaCN chemical hypoxia.