高糖与活性氧对人腹膜间皮细胞p21^(Waf1)表达的影响

Antioxidants reverse high-glucose's effect on p21^(Waf1) expression in human peritoneal mesothelial cells

目的 :研究不同浓度葡萄糖、活性氧 (过氧化氢 )、抗氧化剂 (丙酮酸等 )对体外培养人腹膜间皮细胞(HPMC)细胞周期的影响及其机制。 方法 :以体外培养的HPMC作为研究对象 ,分别给予不同浓度的葡萄糖、过氧化氢、丙酮酸作为刺激因素 ,观察细胞形态 ,用流式细胞仪检测细胞周期改变 ,测定G1期细胞比例。用半定量逆转录多聚酶链反应 (RT PCR)检测细胞周期调控蛋白p2 1Waf1mRNA水平 ,用细胞免疫组化方法检测p2 1Waf1蛋白水平 ,分析细胞周期改变机制。 结果 :高糖、过氧化氢可以引起细胞形态呈肥大、衰老改变 ,细胞周期分析提示G1期细胞比例升高 ,即细胞停滞于G1期 ;加入抗氧化剂 (丙酮酸 )后 ,G1期细胞比例下降。高糖、外源性过氧化氢可以使p2 1表达增加 (基因与蛋白水平 ) ,在高糖培养液中加入抗氧化剂 ,可以使p2 1表达下降。 结论 :高糖、外源性过氧化氢皆可使细胞周期发生停滞 ,且高糖增加外源性过氧化氢的毒性作用 ;高糖的这种作用与内源性活性氧致p2 1Waf1的表达增加有关 。

Objective:High concentration glucose has been associated with peritoneal mesothelial cells damage and fibrosis,and reactive oxygen species(ROS) may implicate in the process of glucose induced mesothelial cells cycle arrest.In this study,we examined the effects of high concentration glucose,small dose exogenous hydrogen peroxide,antioxidants on cell cycle of human peritoneal mesothelial cells(HPMCs). Methodology:Cultured human peritoneal mesothelial cells(HMrSV5) were used in the examination of p21 Waf1 mRNA and protein expression in vitro.Different concentrations of glucose(0 1%?1 35%?3 86%),hydrogen peroxide(0 5 mmol/L,0 1 mmol/L) and the antioxidants(pyruvate,catalase) were applied in the culture of HMrSV5 cells.Morphological changes of HPMC after treatment were studied under light microscopy within 12～24 hours.Flow cytometry was used to measure the proportion of G 1 phase HPMCs.Expression of p21 Waf1 mRNA(one of the Cyclin dependent Kinase Inhibitor,CKI) was determined with semiquantitative reverse transcription polymerase chain reaction (RT PCR),and expression of p21 Waf1 protein measured by immunohistochemistry assisted by a computer analysis software. Results:①Hypertrophy and senescent were observed in HPMC treated by high glucose and by exogenous small dose hydrogen peroxide,with similar morphological changes.②Treatment with high glucose or small dose exogenous hydrogen peroxide resulted in G 1 phase arrest of HPMC with increased proportion of G 1 phase cells,and treatment with both high glucose and hydrogen peroxide resulted in more enhanced G 1 phase arrest.Adding of antioxidants(pyruvate) to high glucose treated or hydrogen peroxide treated HPMC decreased the proportion of G 1 phase arrested cells.③High glucose,small dose hydrogen peroxide increased the expression of mRNA and protein of p21 Waf1 .Adding of antioxidant in high glucose culture medium decreased the expression of p21 Waf1 . Conclusion:High glucose can result in hypertrophy and cell cycle arrest of HPMC.The cell cycle arrest of HPMC in G 1 phase is associated with high glucose's effects on p21 Waf1 expression.These effects of high glucose can be reversed partly by adding of antioxidant.