摘要
目的：建立结核杆菌抗原（Mtb-Ag）再刺激活化的人γδT细胞凋亡模型；应用信号分子阻断剂观察p38及PI3K信号传导途径在Mtb-Ag再刺激诱导γδT细胞凋亡中的作用。方法：用3种浓度Mtb-Ag分别再刺激培养15～25天的Mtb-Ag激活的人T细胞（MtbAT）24小时后，应用Annexin-V-FITC／PI染色流式细胞术（FCM）检测γδT细胞凋亡；用Mtb-Ag（10．0μg／ml）分别再刺激MtbAT3、6、12和24小时后，观察γδT细胞凋亡情况；预先用SB203580（p38途径抑制剂）及LY294002（PI3K途径抑制剂）分别处理MtbAT60分钟，观察Mtb-Ag（10．0μg／m1）再刺激3小时后γδT细胞凋亡情况。结果：与对照组相比，10．0和20．0μg／ml MtbAg均显著诱导γδT细胞凋亡（P〈0．01），凋亡细胞比例分别增加35．63％及38．83％，且此二种浓度MtbAg在诱导γδT细胞凋亡方面无显著性差异（P〉0．05）；与对照组相比，10．0μg/ml Mtb-Ag再刺激MtbAT3、6、12和24小时均可显著诱导γδT细胞凋亡（P〈0．01），凋亡细胞比例在44．21％～51．76％之间；且Mtb-Ag再刺激MtbAT3小时实验组与再刺激MtbAT 24小时实验组相比，在诱导γδT细胞凋亡方面无显著性差异（P〉0．05），后者的凋亡细胞比例仅比前者增加7．55％；Mtb-Ag再刺激诱导γδT细胞凋亡可被SB203580（80．0μmol／L）及LY294002（10．0μmol／L）抑制，凋亡抑制率分别为91．6％及43．1％。结论：采用Mtb-Ag（10．0μg／m1）再刺激活化的γδT细胞3小时的方法，可建立Mtb-Ag再刺激活化的人γδT细胞凋亡模型；信号分子阻断剂的实验证明，p38途径及PI3K途径均参与了Mtb-Ag再刺激已活化γδT细胞凋亡过程。
Objective：To establish the model of apoptosis of activated human γδT cells induced by restimulating with Mycobacterium tuberculosis antigen（Mtb-Ag）. To investigate the roles of p38 and phosphatidylinositol 3 kinase（PDK） pathways in the apoptosis of activated human γδT cells induced by restimualting wih Mtb-Ag. Methods：Mtb-Ag activated human T ceils （MtbAT） were cultured for 15 days to 25 days and restimulated with three concentrations of Mtb-Ag for 24 hours, and the apoptosis of γδT ceils were measnred by flowcytometry（FCM） using Annexin-V-FITC/PI staining. Mtb-AT were restimulating with Mtb-Ag（ 10 μg/ml） for 3, 6, 12 and 24 hours, the apoptosis of γδT cells were detected. Mtb-AT cells were pretreated with SB203580 （ an inhibitor for p38 pathway）, or LY294002（an inhibitor for PI3K pathway） for 60 minutes, and restimulating with Mtb-Ag for 3 hours, the apoptosis of γδT cells were detected. Results：Both 10.0 and 20. 0 μg/ml Mtb-Ag significantly induced the apoptosis of γδT cells（P 〈0. 01 ）, the percentages of apoptosis of γδT cells were 35. 63% and 38. 83%, respectively, and there were not apparent difference between them（P 〉 0. 05）. Compared with control, the apoptosis of γδT cells could be significantly induced by restimulating MtbAT with Mtb-Ag（ 10. 0 μg/ml） for 3, 6, 12 and 24 hours （P 〈 0. 01 ）, the percentages of apoptosis of γδT cells were among 44.21% to 51.76%. There weren＇t profound difference（P 〉0. 05） in the percentages of apoptosis of γδT cells restimulated by Mtb-Ag（ 10. 0 μg/ml） between for 3 hours and for 24 hours, the percentages of apoptosis of the latter is higher than the former about 7. 55%. The apoptosis of γδT cells induced by restimualting wih Mtb-Ag could be inhibited by SB203580（80.0 μmol./L） or LY294002（ 10. 0 μmol/L）, the inhibition rate of apoptosis was 91.6% and 43.1%, respectively. Conclusion：We established the model of apoptosis of activated human γδT cells by means of using Mtb-Ag（ 10. 0 μg/ml） to restimtdate activated γδT cells for 3 hours. The test of inhibitors of signalling molecule suggested the signalling pathways including p38 and PI3K, participated io the apoptosis of activated human γδT cells restimulated by Mtb-Ag.
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2006年第3期203-206,211,共5页
Chinese Journal of Immunology
基金
国家自然科学基金资助项目(30070721)
安徽省高等学校自然科学研究项目(2005KJ3752C)
关键词
ΓΔT细胞
结核杆菌
细胞凋亡
信号转导
γδT lymphocytes
Mycobacterium tuberculosis
Apoptosis
Signal transduction
