Wilms瘤1基因在肾小管上皮细胞转分化中的表达及作用

Expression and effect of Wilms tumor 1 gene on renal tubular epithelial cell transdifferentiation

目的探讨Wilms肿瘤1基因(WT1)在肾小管上皮细胞(TEC)向肌成纤维细胞转分化中的可能作用。方法将体外原代培养TEC分别置含IL-1α(10ng/ml)、IL-1α+抗WT1中和抗体(10μg/ml)的DMEM/F12培养基中培养,观察TEC的形态变化及免疫学特征。采用RT-PCR检测各组TEC中WT1及α-SMA的表达。结果经IL-1α掺入培养5d后,体外培养的TEC形态特征趋于成纤维细胞化,细胞拉长、梭形变.失去原有的呈铺路石样的生长方式。电镜下细胞极性丧失,表面微绒毛消失。正常情况下,成年TEC不表达WT1。经IL-1α掺入培养1d后,TEC重新表达WT1,同时伴有α-SMA的表达;3d后WT1表达消失,呈一过性特征,而α-SMA的表达则随时间的延长而逐渐增强。在培养中掺入抗WT1抗体以中和WT1基因产物后,尽管仍给予IL-1α刺激,TEC大都保持原有特征不变,α-SMA及WT1mRNA仅呈微弱表达。结论高浓度IL-1α可导致TEC向肌成纤维细胞的转分化。在TEC的转分化过程中,WT1基因的重新、一过性表达可能起着重要作用。成年TEC重新获得WT1表达,可能是其发生转分化的内在启动机制。体外中和WT1的基因产物可明显抑制TEC的转分化进程,并可能籍此影响肾脏的纤维化发生。

Objective To observe the process of renal tubular epithelial cell （TEC） transdifferentiation and the expression of Wilms tumor 1 gene（WT1） and to study the effect of WT1 on TEC transdifferentiation induced by IL-1α. Methods TECs were cultured in vitro and WT1 antibody was induced.Confluent ceils were cultured for 5 days in DMEM/F12 media containing IL-1α （10 ng/ml）, with or without 10 μg/ml of mouse anti-WT1 neutralizing antibody, and the ones cultured in DMEM/F12 medium only served as control. TEC morphologic and pbenotype characteristics were observed by light microscope and electron microscope. The expression of α-smooth actin （α-SMA） and WT1 was detected by RT-PCR on day 1, day 2, and day 3 of ceil culture. Results TEC cultured in media with IL-1α （10 ng/ml） for 5 days showed clear morphological and phenotypic changes. Many of them appeared fibroblast-like. Becoming elongated, the transformed ceils showed marked hypertrophyand lost their cobblestone growth pattern. Scanning electron microscopy showed loss of apical-basal epithelial polarity and surface micmvilli on the transformed cells. TEC cultured in media with （10 ng/ml） for 1 day started to re-express WT1 and α-SMA as well. However, WT1 became undetectable on day 3 in a transient pattern, α-SMA＇s expression was enhanced in a time-dependent manner. TEC cultured in media with IL-1α （10 ng/ml） in the presence of 10 μg/ml of mouse neutralizing anti-WT1 antibody had little morphological changes, the WT1 and α-SMA＇s expression clearly decreased in contrast to TEC cultured in media with IL-1α （10 ng/ml）. Conclusions TEC embedded in high concentration of cytokine for long period would induce epithelial-myofibroblast transdifferentiation itself. The transient, re-expression of WT1 could play an important role in epithelial- myofibroblast transdifferentiation. The re-expression of WT1 in adult TEC could be the inner promotor of TEC transdifferentiation.Neutralization of WT1 gene product in vitro could counteract TEC transdifferentiation as well as kidney fibrosis.