摘要
目的 了解肿瘤坏死因子 (TNF α)诱导U937细胞凋亡过程中IκB α蛋白的表达、降解及亚细胞定位 ,并探讨其降解机制。方法 用荧光显微镜观察TNF α诱导细胞凋亡过程中U937细胞内IκB α蛋白的表达及亚细胞定位 ,用流式细胞术分析IκB α蛋白的变化及降解情况 ,并行蛋白酶抑制剂———对甲苯磺酰 L 苯丙氨酸氯甲基甲酮 (TPCK)阻断实验 ,探讨细胞内IκB α蛋白变化的可能机制 ;用流式细胞术分析U937细胞凋亡。结果 ①IκB α分子多呈环状分布于整个胞浆 ,胞核内则无。②TNF α刺激后 ,胞浆中仅见少许IκB α分子 ,胞核内无 ;流式细胞术证实TNF α刺激后一定时间内IκB α蛋白表达水平逐渐降低。③TPCK阻断后 ,IκB α分子仍呈环状分布于整个胞浆 ,胞核内则无。流式细胞术证实在对应时间内其表达明显高于TNF α组。④TNF α诱导的U937细胞凋亡率为 (6 0 .73± 1.6 1) %。结论 ①在TNF α诱导的U937细胞凋亡过程中 ,存在IκB α蛋白降解 ,提示核因子 κB的激活。②IκB α蛋白降解需TPCK敏感的蛋白酶参与。③TPCK敏感的蛋白酶也参与了TNF α诱导U937细胞凋亡。
Objective To investigate the TNF α induced apoptosis of U937 cells, the expression,degradation and subcellular localization of IκB α,and its degradation mechanism. Method Changes and subcellular loca lization of IκB α were observed by fluorescence microscopy, expression and degradation of IκB α protein with N tosyl L phenylalanylchloromethyl ketone (TPCK protease inhibitor ) blocking test and apoptosis of U937 cell by flow cytometry. Results ①immunolfluorescence assay showed that IκB α localized in cytoplasm only. ②The level of IκB α protein was downregulated after TNF α stimulation, flow cytometry also confirmed the downregulation. ③The downregulation of IκB α protein levels in TNF α induced apoptosis was partially inhibited by TPCK.④The apoptosis rate of U937 cells induced by TNF α was (60.73±1.61)%. Conclusion ①Degradation of IκB α protein during TNF α induced apoptosis of U937 cells suggested the activation of NF κB. ②TPCK sensitive protease plays an important role in the degradation of IκB α protein. ③TPCK sensitive protease also involved in the apoptosis of U937 cells induced by TNF α.
出处
《中华血液学杂志》
CAS
CSCD
北大核心
2002年第7期353-355,共3页
Chinese Journal of Hematology
