THE ROLE OF GLOMERULAR ENDTHELIAL CELLS IN THE PATHOGENESIS OF GLOMERULONEPHRITIS.

为探讨肾小球内皮细胞（GEN）在肾小球肾炎发病机制中的作用，采用趋化性实验、北方印迹分析和免疫细胞化学方法，检测了牛GEN在体外产生单核细胞趋化蛋白-1（MCP-1）的情况。通过使用人外周血单核细胞的趋化性测定可检测GEN条件培养基的单核细胞趋化活性。暴露于人重组白细胞介素-1 β（IL-1 β）和佛波醇肉豆蔻酸酯乙酸酯（PMA）显着增加GEN条件培养基的趋化活性。棋盘格分析表明，单核细胞对GEN条件培养基的反应是真正的趋化性。免疫吸附与人MCP-1的单克隆抗体降低了85%的GEN条件培养基的趋化活性。北方印迹分析显示，MCP-1 mRNA由GEN组成性表达，IL-1 β和肿瘤坏死因子-α（TNF-α）在一定剂量和时间内增加MCP-1 mRNA水平 ...更多信息 依赖的方式。PMA还诱导MCP-1 mRNA水平的增加，其被蛋白激酶C抑制剂staurosporine和H-7抑制，而二丁酰环AMP和forskolin的作用最小，表明蛋白激酶C参与。然而，由IL-1 β和TNF-α诱导的MCP-1 mRNA表达被酪氨酸激酶抑制剂genistein抑制，而不是被staurosporine、H-7、或蛋白激酶A抑制剂H-89，提示酪氨酸激酶激活对马槟榔诱导的MCP-1基因表达的重要性。地塞米松对MCP-1 mRNA的表达有一定的抑制作用，但对TNF-α的诱导作用无明显影响。用抗MCP-1单克隆抗体免疫过氧化物酶染色，发现未处理的GEN中有MCP-1蛋白表达，PMA处理后MCP-1蛋白表达增加，与mRNA表达相对应。这些结果证实了GEN在基因和蛋白水平上产生MCP-1以及生物活性，并表明GEN可能通过产生MCP-1参与肾小球肾炎的发展。少

To explore the role of glomerular endothelial cells (GEN) in the pathogenesis of glomerulonephritis, the in vitro production of monocyte chemoattractant protein-1 (MCP-1) by bovine GEN was determined by chemotaxis assay, Northern blot analysis, and immunocytochemistry. Monocyte chemotactic activity of GEN-conditioned media was detectable by a chemotaxis assay using human peripheral blood monocytes. Exposure to human recombinant interleukin-1beta (IL-1beta) and phorbol myristate acetate (PMA) significantly increased the chemotactic activity of GEN-conditioned media. A checcurboard analysis showed that the response of monocytes to GEN-conditioned media was truly chemotactic. Immunoadsorption with a monoclonal antibody to human MCP-1 reduced the chemotactic activity of GEN-conditioned media by 85%. Northern blot analysis revealed that MCP-1 mRNA was constitutively expressed by GEN and that IL-1beta and tumor necrosis factor-alpha (TNF-alpha) increased MCP-1 mRNA levels in a dose- and time … More -dependent manner. PMA also induced an increase in MCP-1 mRNA levels that was suppressed by the protein kinase C inhibitors staurosporine and H-7, whereas dibutyryl cyclic AMP and forskolin had minimal effects, indicating involvement of protein kinase C.However, MCP-1 mRNA expression induced by IL-1beta and TNF-alpha was suppressed by the tyrosine kinase inhibitor genistein, not by staurosporine, H-7, or the protein kinase A inhibitor H-89, suggesting importance of the tyrosine kinase activation on the cytokine-induced MCP-1 gene expression. Dexamethasone had a small inhibitory effect on constitutive and PMA-induced MCP-1 mRNA expression, but no effect on the induction by TNF-alpha.By immunoperoxidase staining with an anti-MCP-l monoclonal antibody, MCP-1 protein was detected in untreated GEN and increased by exposure to PMA,correspondeng to mRNA expression. These results demonstrate the production of MCP-1 by GEN at gene and protein levels as well as bioactivity, and suggest that GEN may participate in the development of glomerulonephritis through the production of MCP-1. Less