IFN-γ和TNF-α联合对MIN6细胞caspase-3活化的影响

Effects of IFN- γ / TNF- α synergism on the activation of caspase-3 in insulinoma cell line MIN6

目的研究细胞因子IFN-γ和TNF-α对小鼠胰岛瘤细胞株MIN6中caspase-3活化的影响。方法用IFN-γ、TNF-α单独或联合刺激MIN6细胞后,Western blot分析比较caspase-3活化的情况,并用免疫细胞化学法检测了其活化产物cleaved caspase-3的产生;酶学比色法测定细胞因子IFN-γ和TNF-α联合作用活化caspase-3的时间依赖性以及MTT法比较caspase-3特异性的抑制剂AC-DEVD预处理前后MIN6细胞活性的变化。结果 IFN-γ或TNF-α单独作用不能活化caspase-3,但两者联合作用24 h后可检测到caspase-3活化裂解片段的产生;Caspase-3的活性随着IFN-γ和TNF-α处理时间的增加而升高,呈现明显的时间依赖性;抑制caspase-3的活化可有效改善MIN6细胞的活性,并且呈剂量依赖性。结论 IFN-γ和TNF-α对MIN6细胞活性的抑制作用与caspase-3的活化密切相关。

The study is aimed to explore the effects of IFN-γ and TNF-α on the activation of caspase-3 in insulinoma cell line MIN6. Western blot was employed to assay the activation of caspase-3, caspase-8 and caspase-9 induced by IFN-γ and TNF-α in MIN6, and immunocytochemistry was used to analyze the production of cleaved caspase-3; the activity of caspase-3 was assayed through enzymology method. On the other hand, MIN6 cells were pretreated with caspase-3 inhibitor AC-DEVD, and then assayed by MTT for cell viability. In contrast to IFN-γ and TNF-α alone, IFN-γ / TNF-α synergism led to an obvious activation of caspase-3 in MIN6 cells and the effects of IFN-γ and TNF-αsynergism on activation of caspase-3 in MIN6 cells was in a time-dependent manner. Furthermore, inhibition of caspase-3 activity led to significantly increased viability of MIN6 cells. Our results suggested that the decrease of MIN6 cells viability by IFN-γ and TNF-α is related to the activation of caspase-3.