论文部分内容阅读
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 changes of α-crystallin in human RPE cells cultured in high glucose in vitro and to compare the effect of osmosis with the normal glucose concentration group. Methods: Human RPE cells (ARPE-19) were cultured and passaged at 5.5 mM D-glucose for 3 weeks and then divided into 3 groups according to culture conditions, namely 5.5 mM glucose, 33 mM glucose, 5.5 mM glucose + 27.5 mM mannose Alcohol, and then incubated for 12h, 24h, 48h after total protein and RNA extraction. Furthermore, αA crystallin, αB crystallin and bax, bcl-2 were detected by Western blotting. The mRNA levels of αA- and αB- were detected by RT-PCR. Results: αA crystallin and αB crystallin were significantly upregulated in high glucose condition compared with normal glucose concentration group, while osmotic change had no significant effect on protein expression. Under the condition of high glucose, the expression of bcl-2 decreased significantly, while the expression of bax increased. The changes of mRNA of αA- and αB- were consistent with the protein level. CONCLUSION: Both αA- and αB- crystallin proteins in human retinal pigment epithelial cells are obviously upregulated in high glucose culture environment.