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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。
Objective To study the effect of cytokines IFN-γ and TNF-α on the activation of caspase-3 in mouse pancreatic cancer cell line MIN6. Methods MIN6 cells were stimulated with IFN-γ and TNF-α alone or in combination. The activation of caspase-3 was detected by Western blot and the cleaved caspase-3 was detected by immunocytochemistry. The time dependent activation of caspase-3 by cytokines IFN-γ and TNF-α was measured and MTT assay was used to compare the changes of MIN6 cell activity before and after pretreatment with caspase-3 inhibitor AC-DEVD. Results Caspase-3 could not be activated by IFN-γ or TNF-α alone. However, the cleavage of caspase-3 was detected 24 h after treatment with IFN-γ or TNF-α. The treatment time increased with time, showing a significant time-dependent; inhibition of caspase-3 activation can effectively improve the activity of MIN6 cells in a dose-dependent manner. Conclusion The inhibitory effect of IFN-γ and TNF-α on the activity of MIN6 cells is closely related to the activation of caspase-3.