摘要
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.
出处
《中华肾脏病杂志》
CAS
CSCD
北大核心
2005年第8期448-452,共5页
Chinese Journal of Nephrology