酸性成纤维细胞生长因子诱导大鼠肾小管上皮细胞转分化的作用及机制

Acid fibroblast growth factor induces renal tubular epithelial-myofibroblast transdifferentiation in vitro

目的 探讨酸性成纤维细胞生长因子 (AcidfibroblastgrowthfactoraFGF)在大鼠肾小管上皮细胞株 (NRK)转分化中的作用 ,为肾小管 间质纤维化的防治提供理论依据。方法 在NRK细胞的培养基内加入不同剂量的重组人aFGF ,并在Ⅰ型胶原处理过及未处理的玻璃表面 ,无血清培养 6d。使用透射电镜、免疫细胞化学对肾小管上皮细胞向肌成纤维细胞形态及表型的转变进行评估。结果 不同剂量的aFGF(1、1 0、50ng/ml)处理后的NRK细胞形态和表型发生不同程度的改变 :体积增大、失去尖端 基底极性和微绒毛、细胞延长、具有浸润特征的前后向双末端极性、出现肌动蛋白中间丝结构。免疫组化和Westernblot分析显示加入aFGF后角蛋白表达减少 ,有肌成纤维细胞的特异性标志物α 平滑肌动蛋白 (α SMA)的表达。α SMA阳性细胞的比例与aFGF的浓度呈正相关 ,亲和抗aFGF抗体可消除这种转分化现象。加入aFGF后 ,在玻璃表面α SMA阳性细胞数 (34 4± 0 0 90 ) %较Ⅰ型胶原处理培养表面α SMA阳性细胞数 (74 6± 0 1 0 1 ) %有显著差异 (P <0 0 5)。结论 aFGF在促进肾小管上皮细胞向肌成纤维细胞转分化的过程中有重要的意义 ,该过程亦与细胞的生长环境有关 。

Objective To investigate the possible mechanisms of acid fibroblast growth factor (aFGF) in the phenotypic transformation of rat renal tubular epithelial cells line (NRK) in vitro . Methods Normal rat renal tubular epithelial line (NRK) were cultured for 6 d on the plate with or without collagen typeⅠ coating and in the absence or presence of aFGF at different doses. Transdifferentiation of tubular cells into myofibroblasts was assessed with electron microscopy and by detecting the expression of α smooth muscle actin (α SMA) and cytokeratin. Results NRK cells cultured on collagen coated or not coated plates showed a normal epithelial morphology. But the cells cultured with 50 ng/ml aFGF on collagen coated plates showed profound changes, including elongated and enlarged cell body, loss of apical basal polarity and microvilli, formation of new front end back end polarity, appearance of actin microfilaments and features of invasiveness. These morphological changes were companied by phenotypic changes. Immunohistochemical staining showed that the addition of aFGF to subconfluent cell culture caused the loss of the epithelial marker cytokeratin and novo expression of α SMA. The transformed cells showed a fibroblast like reorganization of actin fibers along with the cell axis. The percentage of cells expressing α SMA was increased along with concentrations of aFGF in a dose dependent manner, which was inhibited by the addition of anti aFGF antibody. Compared with the cells growing on plates without coating, cell cultured on collagen coated plates showed a significance increase ( P <0.05) in the percentage of cells expressing α SMA in response to aFGF. Conclusion aFGF is a key mediator that regulates, in a dose dependent manner, the transdifferentiaton of renal tubular epithelial cells into α SMA + myofibroblasts. This transdifferentiation is markedly enhanced by growth on collagen type Ⅰ. These findings indicate a novel pathway that may contribute to renal tubulointerstitial fibrosis.