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 huma...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.展开更多
To evaluate the protective effects and mechanism of action of oxymatrine (OM) on the experimental fulminant hepatitis (FH) and early hepatocyte apoptosis in mur ine liver tissue Methods Fulminant hepatitis mice wer...To evaluate the protective effects and mechanism of action of oxymatrine (OM) on the experimental fulminant hepatitis (FH) and early hepatocyte apoptosis in mur ine liver tissue Methods Fulminant hepatitis mice were induced by injecting lipopolysaccharide (LPS) intr aperitoneally (ip) in galactosamine (GalN) sensitized mice Two separate experim ents were designed, including saline control group, fulminant hepatitis group an d oxymatrine pretreated group (50?mg/kg, intraperitoneally, bid×3 days) The leve ls of serum tumor necrosis factor alpha (TNFa) in mice from two experiments were determined at 5 hour and 7 5 hour after injecting galactosamine/lipopolysacc haride Mouse liver samples at 5 hour time point were obtained for in situ end labeling (ISEL) staining and ultrastructural observation of apoptotic cells und er transmission electron microscope (TEM) Liver samples at 7 5 hour time poi nt were taken for hematoxylin eosin (HE) staining and immunohistochemical stain ing of Fas and its ligand (FasL) Results As compared with the fulminant hepatitis group, the levels of serum tumor necros is factor alpha in mice from the OM pretreated group at 5 hour and 7 5 hour t ime point were all significantly decreased ( P 【0 05 and P 【0 01 respecti vely) Hepatocyte apoptosis in mice at 5 hour time point was significantly inh ibited ( P 【0 01) Both the degree of liver injury and the degree of Fas and Fas ligand expression in the OM pretreated group were reduced remarkably ( P 【0 01 and 0 05 respectively) when compared with the saline control group Conclusions Oxymatrine protects mice from fulminant hepatitis induced by GalN/LPS and may bl ock hepatocyte apoptosis and subsequent necrosis through downregulating the prod uction of serum tumor necrosis factor alpha and the expression of Fas and Fas li gand in liver tissue展开更多
文摘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.
文摘To evaluate the protective effects and mechanism of action of oxymatrine (OM) on the experimental fulminant hepatitis (FH) and early hepatocyte apoptosis in mur ine liver tissue Methods Fulminant hepatitis mice were induced by injecting lipopolysaccharide (LPS) intr aperitoneally (ip) in galactosamine (GalN) sensitized mice Two separate experim ents were designed, including saline control group, fulminant hepatitis group an d oxymatrine pretreated group (50?mg/kg, intraperitoneally, bid×3 days) The leve ls of serum tumor necrosis factor alpha (TNFa) in mice from two experiments were determined at 5 hour and 7 5 hour after injecting galactosamine/lipopolysacc haride Mouse liver samples at 5 hour time point were obtained for in situ end labeling (ISEL) staining and ultrastructural observation of apoptotic cells und er transmission electron microscope (TEM) Liver samples at 7 5 hour time poi nt were taken for hematoxylin eosin (HE) staining and immunohistochemical stain ing of Fas and its ligand (FasL) Results As compared with the fulminant hepatitis group, the levels of serum tumor necros is factor alpha in mice from the OM pretreated group at 5 hour and 7 5 hour t ime point were all significantly decreased ( P 【0 05 and P 【0 01 respecti vely) Hepatocyte apoptosis in mice at 5 hour time point was significantly inh ibited ( P 【0 01) Both the degree of liver injury and the degree of Fas and Fas ligand expression in the OM pretreated group were reduced remarkably ( P 【0 01 and 0 05 respectively) when compared with the saline control group Conclusions Oxymatrine protects mice from fulminant hepatitis induced by GalN/LPS and may bl ock hepatocyte apoptosis and subsequent necrosis through downregulating the prod uction of serum tumor necrosis factor alpha and the expression of Fas and Fas li gand in liver tissue
