摘要
Objective To detect if Fas is expressed in human renal interstitial fibroblasts (hRIFs) and apoptosis of hRIFs can be induced by specific anti-Fas antibody. Methods hRIFs were cultured from isolated papillae of human kidney, and identified by morphologic examination, assay of antigenic components and culture in D-valine selective medium. Fas expression in normal hRIFs was detected by RT-PCR and immunocytochemistry staining. After hRIFs were incubated with interferon gamma (γ-IFN 500 U/ml, 1000 U/ml, 1500 U/ml and 2000 U/ml, respectively) for 48 hours, Fas expression was determined by Northern blot, Western blot and flow cytometry. hRIFs pre-stimulated with γ-IFN (500 U/ml, 48 hours) were incubated with anti-Fas antibody (IgM, 0.5 μg/ml) for 12 hours. And apoptosis was identified by morphologic examination, DNA ladder assay and flow cytometry.Results The cultured hRIFs showed a shuttle-like shape and were positively stained by labeled anti-vimentin antibody but negatively stained by anti-epithelial membrane antibody. They could not grow in the D-valine selective medium and died partly in a week. Fas mRNA and protein were expressed in normal hRIFs and markedly upregulated by stimulation with γ-IFN. Apoptosis in γ-IFN pre-stimulated hRIFs was induced by anti-Fas antibody, showing cell nuclear shrinkage and condensation in morphologic feature, internucleosomal DNA fragmentation in DNA ladder assay and a pick of hypo-diploid nuclei by flow cytometry. Conclusion Fas is normally expressed in hRIFs and can be markedly upregulated by γ-IFN. Anti-Fas antibodies can induce apoptosis of hRIFs pre-stimulated with γ-IFN.
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 。
