病理性损伤因素对肾小管上皮细胞表型转化的影响

目的 :观察低血清、高糖、高蛋白刺激下肾小管上皮细胞向间叶性细胞的表型转化 ,探讨损伤的肾小管上皮细胞在肾小管间质纤维化中的作用。方法 :以体外培养的人近端肾小管上皮细胞为对象 ,分别用含低血清(0 .2mol·L-1小牛血清 )、高葡萄糖 (4.5 g·L-1)、高白蛋白 (15 g·L-1)的培养液培养 7d ,透射电镜检测细胞形态变化 ;免疫组化和Westernblot检测细胞表型改变 ,包括CK、Vimentin、α SMA、Ⅰ和Ⅲ型胶原 ,原位杂交检测Ⅰ型胶原mRNA表达 ,同时检测细胞TGFβ1蛋白表达。 结果 :低血清、高糖、高蛋白直接刺激下 ,肾小管上皮细胞出现明显形态学改变 ,包括细胞变为长梭形 ;透射电镜下细胞内线粒体明显减少 ,粗面内质网明显增加 ,并出现actin样微丝。免疫组化染色和Westernblot显示CK表达明显减弱 ,Vimentin、Ⅰ、Ⅲ型胶原、α SMA和TGFβ1表达增强。原位杂交显示Ⅰ型胶原mRNA表达增强。结论 :低血清、高糖、高蛋白可直接引起人近端肾小管上皮细胞TGFβ1表达增加 ,并发生向间叶性细胞的表型转化

The phenotypic transformation of renal tubular epithelial cells affected by pathological stimuli

OBJECTIVE: To investigate the effects of low bovine serum, high glucose and high albumin on the phenotypic transformation of cultured renal tubular epithelial cells to mesenchymal cells. METHODS: The human kidney proximal tubular cells were cultured for 7 days in the presence of low bovine serum (0.2 mol.L-1), high glucose (4.5 g.L-1) and high albumin (15 g.L-1), and morphological changes were assessed by electronic microscopy, immunohistochemistry stain and Western blot to show the phenotypic changes, including the expressions of cytokeratin, vimentin, collagen I and III, alpha-SMA, and TGF beta 1. The expression of collagen I mRNA was assessed by in situ hybridization. RESULTS: Renal tubular epithelial cells cultured in low bovine serum, high glucose and high albumin, showed obvious morphologic changes, including elongated shape, reducing of microvillus and mitochondria, increasing of rough endoplasmic reticulum, and the appearance of actin microfilaments. Immunohistochemistry stain and Western blot showed the low expression of cytokeratin and high expression of vimentin, collagen types I and III, alpha-SMA, and TGF beta 1. The increasing expression of collagen I mRNA was assessed by in situ hybridization. CONCLUSION: Lower bovine serum, high glucose and high albumin can induce the expression of TGF beta, and lead to the phenotypic transformation of tubular epithelial cells to mesenchymal cells.