Fas、NF-κB及Caspases在TNFα介导的微血管内皮细胞凋亡中的作用

Effects of Fas, NF-κB and caspases on microvascular endothelial cell apoptosis induced by TNFα

目的 :研究肿瘤坏死因子α(TNFα)引起微血管内皮细胞凋亡的特点及Fas(即CD95抗原 )、NF -κB和caspases在此种凋亡中的作用。方法 :用TUNEL、流式细胞仪等方法测定TNFα引起体外培养的大鼠肺微血管内皮细胞 (下称内皮细胞 )凋亡。用Northernblot、Fas抗体测定TNFα对内皮细胞Fas表达的影响及Fas在内皮细胞凋亡中的作用。用Westernblot、免疫组化测定TNFα作用后凋亡蛋白酶caspase - 8、caspase- 3的活化与表达。结果 :细胞生长曲线显示TNFα抑制内皮细胞增殖 ,并呈剂量依赖关系。TUNEL法测凋亡百分率为 14.0 %± 3.1% ,流式细胞仪测凋亡百分率为 13.1%。NF -κB抑制剂吡咯烷二硫氨基甲酸盐 (APDC)与TNFα共同作用后 ,细胞凋亡增加。TNFα作用后内皮细胞的Fas表达增高 ,用Fas抗体与TNFα共同作用 ,细胞凋亡增加。TNFα作用下caspase- 8活化显著增加 ,caspase - 3表达增多。caspase - 3抑制剂与TNFα共同作用 ,细胞凋亡减少。结论 :大剂量TNFα (5× 10 6U/L)能够引起一定量的体外培养的内皮细胞发生凋亡。NF -κB有抗内皮细胞凋亡的作用。TNFα上调内皮细胞Fas表达 ,后者参与内皮细胞凋亡。TNFα所引起的内皮细胞凋亡与caspase- 8及caspase -

AIM:To study the apoptotic effect of TNFα on rat pulmonary microvascular endothelial cells (PMVEC)and the role of Fas, NF-κB in its mechanism. METHODS: Apoptosis of PMVEC was analyzed and quantitated with TUNEL, flow cytometer. Northern blot was applied to assess the influence of TNFα on PMVEC Fas expression. Fas antibody was used to investigate the apoptotic effect of Fas on PMVEC. Activation of caspase-8 was examined by Western blot. Expression of caspase-3 was analyzed with histo-immunochemical staining. RESULTS:Growth curve showed that TNFα suppressed endothelial cell proliferation in a dose-dependent manner. After treatment with 5×10 6 U/L TNFα, apoptotic rate was 14.0%±3.1% detected with TUNEL, and 13.1% with flow cytometer. When the cells were co-cultured with TNFα and APDC, an NF-κB inhibitor, less cells were viable and more cells were positively stained with TUNEL. Fas expression in PMVEC was elevated after TNFα treatment. Co-culturing with Anti-Fas antibody aggravated PMVEC apoptosis. Caspase-8 activity and caspase-3 expression was elevated. Caspase-3 inhibitor significantly ameliorated PMVEC apoptosis. CONCLUSION: Large dose of TNFα (5×10 6 U/L) can induce apoptosis in rat PMVEC. NF-κB has an anti-apoptotic effect in PMVEC. TNFα up-regulates Fas expression in PMVEC, and the latter takes a part in apoptosis. Caspase-8 and caspase-3 are involved in PMVEC apoptosis induced by TNFα.