CAJ | 학술논문

目的通过观察高糖是否引起小鼠足细胞发生上皮-间叶细胞转分化现象,探讨糖尿病肾小球损伤的可能机制.方法以小鼠永生化足细胞株为研究对象,予不同浓度葡萄精(12.5、25、50 mmol/L)处理该细胞36 h.并设低糖(5.6 mmol/L)和甘露醇(50 mmol/L)处理组为对照组.采用蛋白免疫印迹和间接免疫荧光染色方法检测α平滑肌肌动蛋白(α-SMA)、迁连蛋白(FN)、CD_2相关蛋白(CD2AP)和Wilms'肿瘤1基因(WT-1)蛋白的表达.结果低糖(5.6 mmol/L)和甘露醇(50 mmol/L)处理条件下小鼠足细胞表达WT-1、CD2AP.基本小表达α-SMA和FN;而予不同浓度葡萄精处理36 h后,足细胞中α-SMA和FN表达水平呈剂量依赖性上调(P<0.05).间接免疫荧光染色结果也显示,与低糖组相比,高精组中α-SMA阳性的足细胞比例显著增加(P<0.05).同时,蛋白免疫印迹结果还表明高糖可呈剂量依赖性方式下调WT-1和CD2AP的表达(P<0.05).结论在高糖条件下,足细胞发生向间叶细胞表型的转分化可能是引起足细胞功能失调.进而引起糖尿病肾小球损伤发生的作用机制之一.
목적 통과관찰고당시부인기소서족세포발생상피-간협세포전분화현상,탐토당뇨병신소구손상적가능궤제.방법 이소서영생화족세포주위연구대상,여불동농도포도정(12.5、25、50 mmol/L)처리해세포36 h.병설저당(5.6 mmol/L)화감로순(50 mmol/L)처리조위대조조.채용단백면역인적화간접면역형광염색방법검측α평활기기동단백(α-SMA)、천련단백(FN)、CD_2상관단백(CD2AP)화Wilms'종류1기인(WT-1)단백적표체.결과 저당(5.6 mmol/L)화감로순(50 mmol/L)처리조건하소서족세포표체WT-1、CD2AP.기본소표체α-SMA화FN;이여불동농도포도정처리36 h후,족세포중α-SMA화FN표체수평정제량의뢰성상조(P<0.05).간접면역형광염색결과야현시,여저당조상비,고정조중α-SMA양성적족세포비례현저증가(P<0.05).동시,단백면역인적결과환표명고당가정제량의뢰성방식하조WT-1화CD2AP적표체(P<0.05).결론 재고당조건하,족세포발생향간협세포표형적전분화가능시인기족세포공능실조.진이인기당뇨병신소구손상발생적작용궤제지일.
Objective To investigate the possible mechanism of glomerular injury in diabetes mellitus by determining whether epithelial-mesenchymal transition (EMT) is caused by high glucose in mice podocytes. Methods Using mice glomerular podocyte cell line as an in vitro system, podocytes were incubated with glucose(12.5 mmol/L, 25 mmol/L, 50 mmol/L) and mannitol (50 mmol/L) for 36 hours. Then the cells were collected and expression of alpha-smooth muscle actin(α-SMA), fibronectin (FN), CD2 associated protein (CD2AP) and Wilms' tumor 1 gene (WT-1) was detected by Western blot and indirect immunofluorescence staining. Results Under low glucose (5.6 mmol/L) and mannitol (50 mmol/L) condition, there were high expression of CD2AP and WT-1, and low expression of α-SMA and FN in mice podocytes. After 36 hours treatment with high glucose (12.5 mmol/L), the expression of α-SMA and FN in podocytes was significantly increased, and the expression of α-SMA and FN was further up-regulated with the increase of glucose dosage (25, 50 mmol/L). The indirect immunofluorescence staining revealed the similar result, and the percentage of positive α-SMA cells was also increased compared with low glucose and mannital group (P<0.05). Meanwhile, Western blot showed that high glucose could down-regulate the expressions of CD2AP and WT-1 in a dose-dependent manner. Conclusion EMT may be a potential pathway leading to podocyte dysfunction and glomerular injury under high glucose conditions.