病理性因素对体外肾小管上皮细胞表型转化的影响

Influence of pathologic factors on phenotypic transformation of renal tubular epithelial cells in vitro

目的通过低血清、高糖、高白蛋白的刺激,观察肾小管上皮细胞的表型变化。方法以体外培养的人近端肾小管上皮细胞系HKC为对象,分别用低血清(2％小牛血清)、高糖(25 mmol/L)、高白蛋白(1.5 g/L)刺激,用透射电镜检测细胞形态变化;用免疫组织化学(组化)检测角蛋白(CK)、波形蛋白(vimentin)、α-平滑肌肌动蛋白(SMA)、I型胶原和转化生长因子(TGF)-β1的蛋白表达;Western blot进一步检测I型胶原蛋白表达的动态变化;细胞原位杂交检测I型胶原基因表达。结果在低血清、高糖、高白蛋白直接作用下,肾小管上皮细胞形态变化明显,包括呈梭形改变。透射电镜下细胞表面微绒毛及细胞内线粒体明显减少,粗面内质网明显增加。免疫组化染色显示CK减弱,vimentin、α-SMA、I型胶原和TGF-β1增强。Western blot显示I型胶原表达增加,且与损伤程度正相关。原位杂交显示I型胶原基因表达增加。结论在低血清、高糖、高白蛋白直接作用下,体外培养的正常人近端肾小管上皮细胞系HKC发生了上皮向问叶的表型转化。

Objective To study the effects of high albumin, high glucose and low bovine serum on the phenotypic transformation of renal tubular epithelial cells. Methods The normal human kidney proximal tubular eel] line (HKC) was cultured for 30 days in the presence of high albumin (1.5 g/L), high glucose(25 mmol/L) and low bovine serum(2% ) . Morphological changes were observed by electronic microscopy. Immunohistochemistry stain was used to examine the expression of cytokeratin, vimentin, a-SMA, collagen Ⅰ and TGF-pl protein. Western blot was applied to further detect the process of collagen I protein expression, and in situ hybridization was used to examined the expression of collagen Ⅰ gene. Results Renal tubular epithelial cells cultured in high albumin, high glucose and low bovine serum showed obvious morphologic changes, including elongated shape, decrease of microvilli and mitochondria, and increase of rough endoplasmic reticulum under electronic microscopy. Immunohistochemistry stain revealed the reduction of cytokeratin, and enhancement of vimentin, α-SMA, TGF-β1 and collagen Ⅰ. Western blot demonstrated that the expression of collagen Ⅰincreased in a time-dependent manner, and in situ hybridization showed that collagen type Ⅰ mRNA increased as well. Conclusion High albumin, high glucose and low bovine serum induce phenotypic transformation of renal tubular epithelial cells into mesanchymal cells.