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目的观察肿瘤坏死因子-α(tumor necrosis factor, TNF-α)能否促人腹膜间皮细胞表型向成纤维细胞表型转化,并同时位有相应细胞功能的改变.方法 TNF-α刺激体外培养的人腹膜间皮细胞株(HMrSV5,此细胞株由Pierre RONCO教授馈赠),用相差显微镜观察人腹膜间皮细胞(human peritoneal mesothelial cels, HPMCs)形态的变化 ;用免疫细胞化学方法观察HPMCs细胞角蛋白8,细胞角蛋白18和波形蛋白表达的变化;用RT -PCR方法观察HPMCs的MMPs及TIMPs mRNA表达的变化;用ELISA方法检测MMP-9和Ⅰ型胶原的合成.结果 TNF-α(1-10 ng/ml)可促部分HPMCs(约50%-60%)的细胞形态由原来的多角形转变为细胞两端呈针尖样改变的长梭形,即典型的成纤维细胞样形态.TNF-α(1-10 ng/ml)可促形态转变的HPMCs细胞角蛋白8和细胞角蛋白18由原来的强阳性转为阴性;波形蛋白表达保持强阳性 .此改变符合成纤维细胞表面抗原的特征性分布.TNF-α(1-10 ng/ml)刺激HPMCs4小时后 ,HPMCs的MMP9 mRNA表达明显上调;TIMP-1、TIMP-2 mRNA表达轻微下调.另外,ELISA检测结果发现TNF-α(0.5-10 ng/ml)刺激HPMCs24小时后,MMP-9和Ⅰ型胶原的合成显著增加.结论 TNF-α刺激24小时后,部分HPMCs的表型向成纤维细胞表型转变;同时MMP-9和Ⅰ型胶原的合成明显增加.这些变化有利于促进腹膜细胞外基质的重构和纤维化的形成.

Objective To investigate if tumor necrosis factor-α (TNF-α) could induce phenotypic transformation or the associated cell function changes of human peritoneal mesothelial cells (HPMCs).Methods All tests were performed on the human peritoneal mesothelial cell line (HMrSV5, kindly donated by Prof. Pierre RONCO). Morphological changes of HPMCs were observed by phase contrast microscopy. The expressions of cytokeratin 8, cytokeratin 18 and vimentin were detected by immunocytochemistry. mRNA expressions of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) were detected by semiquantitative reverse transcription polymerase chain reaction (RT-PCR). Production of MMP-9 and type Ⅰ collagen was measured by enzyme-linked immunosorbent assays. Results TNF-α (1-10ng/ml) induced morphological changes in about 50%-60% of human peritoneal mesothelial cells (HPMCs). The original polygonal cobblestone monolayer was changed into elongated, spindle-shape characteristic of fibroblasts. The original strong positive expression of cytokeratin 8 and cytokeratin 18 was changed in all morphologically changed HPMCs to negative, but the expression of vimentin maintained positive. By 4-hour treatment with TNF-α (1-10ng/ml), mRNA expression of MMP9 was significantly up-regulated, and mRNA expression of TIMP-1 and TIMP-2 were down-regulated. We also observed that the production of MMP-9 and type Ⅰ collagen increased significantly after 24-hour treatment with TNF-α. Conclusion 24-hour treatment with TNF-α, produced a partial transformation of HPMCs into the fibroblast phenotype. TNF-α treatment of HPMCs up-regulated the synthesis of MMP-9 and type Ⅰ collagen, which may facilitate peritoneal extracellular matrix remodeling and fibrogenesis.