α-晶状体蛋白在高糖培养人RPE细胞中的表达机制研究

The Expression and Mechanism ofα- crystalline in High Glucose Cultured Human RPE Cells

目的:研究高糖体外培养环境下人RPE细胞的α晶状体蛋白的表达变化,并排除渗透影响因素下与正常糖浓度组进行比较。方法:对人RPE细胞(ARPE-19)在D-葡萄糖为5.5mM条件下培养和传代3周,然后按培养条件分为3组,即5.5mM葡萄糖,33mM葡萄糖,5.5mM葡萄糖+27.5mM甘露醇,再分别培养12h,24h,48h后进行总蛋白和RNA提取。进而通过Western blotting对αA晶状体蛋白,αB晶状体蛋白以及凋亡相关蛋白bax,bcl-2进行检测,并通过RT-PCR检测αA-,αB-两种晶状体蛋白的mRNA水平。结果:相对于正常糖浓度组,高糖环境下αA晶状体蛋白,αB晶状体蛋白均表达明显上调,渗透变化对蛋白表达没有明显影响。高糖培养环境下,随着时间延长,bcl-2表达量明显下降,而bax表达量呈上升趋势,αA-,αB-两种晶体蛋白的mRNA变化趋势与蛋白水平一致。结论:高糖培养环境下人视网膜色素上皮细胞的αA-,αB-两种晶状体蛋白均明显表达上调。

Objective： To study the expression and related mechanism of α-erystallines in high glucose cultured human RPE cells. Methods： Human RPE cells （ARPE-19） were cultured and passaged in medium with 5.5mM D-Glucose for 3 weeks, then divided into three groups according to culture conditions, namely, 5.5mM glucose, 33raM glucose, 5.5mM glucose added 27.5mM mannitol. After 12h, 24h, 48h culute, the total protein and total RNA was extracted, and the expression of αA-crystalline, αB-crystalline, bax, bcl-2 were detected by western blot, then the mRNA of αA-crystalline, αB-crystalline were detect with RT-PCR. Results： Contrary to normal glucose concentrations, αA-erystalline, αBcrystalline protein expression was significantly increased in high glucose. Permeability changes had no effect on protein expression, along with the time, however, the expression of bcl-2 was significantly decreased in high glucose, while bax had an upward trend. RT-PCR results had a similar trend with proteins expression. Conclusions： Cultured in high glucose, αA-crystalline and ~xB-crystalline are significantly up-regulated in human RPE cells, and associated with the expression change of apoptosis-related protein. Bcl-2, bax may play a role in RPE cells＇ α-crystalline expression change in high glucose.