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目的 检测人肾间质成纤维细胞 (hRIFs)Fas表达并以抗Fas抗体诱导hRIFs凋亡。方法 分离人肾肾乳头组织培养hRIFs,以细胞形态、免疫细胞化学染色和右旋缬氨酸选择性培养基培养作细胞鉴定 ;采用RT PCR、免疫细胞化学染色检测正常hRIFsFas表达 ,γ干扰素 (γ IFN ,50 0U/ml、10 0 0U/ml、150 0U/ml和 2 0 0 0U/ml)与hRIFs孵育 4 8小时后 ,采用Northern杂交、Western印迹和流式细胞术检测Fas表达 ;经γ IFN预处理 (50 0Uμ/ml,4 8小时 )的hRIFs ,加入抗Fas抗体 (IgM ,0 5μg/ml)作用 12小时 ,以形态学、DNA电泳和流式细胞术观察细胞凋亡。结果 培养细胞呈梭形 ,波形蛋白 (+ ) ,上皮细胞膜抗原 ( ) ,选择性培养基内逐渐死亡 ;正常hRIFs有FasmRNA和蛋白质表达 ,γ IFN可明显上调其表达水平 ;抗Fas抗体可引起高表达Fas的hRIFs核固缩、核断裂 ,DNA电泳呈现梯形结构 ,流式细胞术呈现亚二倍体凋亡细胞峰。结论 Fas表达于hRIFs且可被γ IFN显著上调 ,抗Fas抗体能够诱导高表达Fas的人肾间质成纤维细胞凋亡
Objective To detect Fas expression in human renal interstitial fibroblasts (hRIFs) and induce apoptosis of hRIFs with anti-Fas antibody. Methods HRIFs were isolated from human kidney and papillary tissue and cultured for cell identification by cell morphology, immunocytochemistry, and dextran-selective medium. RT-PCR and immunocytochemistry were used to detect the expression of normal hRIFsFas and interferon-γ ( After incubated with hRIFs for 48 hours, γ IFN (50 μl/ml, 100 μl/ml, 100 μl/ml, 150 μl/ml, and 200 μl/ml) was used to detect Fas expression by Northern blot, Western blot, and flow cytometry. hRIFs pretreated with γ IFN (50 μl/ml, 48 hours) were treated with anti-Fas antibody (IgM, 0 5 μg/ml) for 12 hours. Cell apoptosis was observed by morphology, DNA electrophoresis, and flow cytometry. Results The cultured cells were fusiform, vimentin (+), epithelial membrane antigen (), and gradually died in selective medium; normal hRIFs had Fas mRNA and protein expression, γ IFN could significantly upregulate their expression level; anti-Fas antibody could cause high The nuclear condensation and nuclear fragmentation of hRIFs expressing Fas were observed. DNA electrophoresis showed a trapezoidal structure. Flow cytometry showed hypodiploid apoptotic cell peaks. Conclusion Fas is expressed in hRIFs and can be significantly upregulated by γ IFN. Anti-Fas antibody can induce apoptosis of human renal interstitial fibroblasts with high expression of Fas.