论文部分内容阅读
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ｈＲＩＦｓ Ｆａｓ表达；不同γ干扰素浓度（γ－ＩＦＮ，５００ Ｕ／ｍｌ、１０００ Ｕ／ｍｌ、１５００／ｍｌ和２ ０００ Ｕ／ｍｌ）与ｈＲＩＦｓ孵育４８ ｈ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（５００ Ｕ／ｍｌ，４８ ｈ）的 ｈＲＩＦｓ，加入抗Ｆａｓ抗体（ＩｇＭ，０．５ｍｇ／ｍｌ）作用１２ｈ，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 Ｆａｓ ｍＲＮＡ和蛋白表达，γ－ＩＦＮ可明显上调其表达水平，直接免疫荧光流式细胞术测定未加 γ－ＩＦＮ的 ｈＲＩＦｓ中 Ｆａｓ表达阳性细胞占 ５．９１％，上述浓度的γ－ＩＦＮ作用后依次为 ５９．４４％、６９．３９％、７０．０６％和 ７５．４７％，呈剂量依赖性增强。抗Ｆａｓ抗体可引起高表达Ｆａｓ的ｈＲＩＦ?
Objective To detect the expression of Fas in human renal interstitial fibroblasts (hRIFs) and induce the apoptosis of hRIFs with anti-Fas antibody. Methods Human renal papilla tissue culture hRIFs were isolated and identified by cell morphology, immunocytochemistry and dextrose-selective culture. The expression of Fas in normal hRIFs was detected by RT-PCR and immunocytochemical staining. The cells were incubated with hRIFs for 48 h at different concentrations of IFN-γ (γ-IFN, 500 U / ml, 1000 U / ml, 1500 / ml and 2000 U / ml) After Northern blotting, Western blotting and flow cytometry were used to detect Fas expression. HRIFs pretreated with γ-IFN (500 U / ml, 48 h) were treated with anti-Fas antibody (IgM, 0.5 mg / ml) for 12 h. Morphological, DNA electrophoresis and flow cytometry were used to observe the apoptosis. Results The cultured cells were fusiform, vimentin (+), epithelial membrane antigen (-) and gradually died in selective medium. The expression of Fas mRNA and protein in normal hRIFs was up-regulated. The expression of Fas mRNA in hRIFs was up-regulated by direct immunofluorescence flow cytometry. The percentage of Fas positive cells in the hRIFs without IFN-γ was 5.91% The effects of IFN were 59.44%, 69.39%, 70.06% and 75.47%, respectively, in a dose-dependent manner. Anti-Fas antibodies can cause high expression of Fas hRIF?