蛋白激酶Cθ在γδT细胞表达L-选择素中的调控作用  

Effect of protein kinase CO on the regulation of L-selectin expression of human γδT cells

作　　者：武文娟[1] 耿英华[3] 唐洁[2] 李柏青[2] 

机构地区：[1]蚌埠医学院生物化学与分子生物学教研室,233030 [2]安徽省感染与免疫重点实验室,免疫学教研室 [3]蚌埠医学院附属医院血液科

出　　处：《中华微生物学和免疫学杂志》2009年第2期146-150,共5页Chinese Journal of Microbiology and Immunology

基　　金：安徽省自然科学基金项目（050430603）;安徽省教育厅自然科学基金项目（KJ20078369ZC）;安徽省高等学校青年教师科研资助计划项目（2007jq1132）;蚌埠医学院科研基金项目（BY0508）

摘　　要：目的探讨蛋白激酶Cθ（PKCθ）信号转导途径在γδT细胞表达L-选择素（CD62L）中的调控作用。方法用结核分枝杆菌抗原（Mtb-Ag）刺激人外周血单个核细胞（PBMC）并培养6～8d，获得富含γδT细胞的结核分枝杆菌活化T细胞（MtbAT），作为γδT细胞来源，分别加佛波醇酯（PMA）、PMA＋离子霉素（IMN）培养3、6、12、24h，或用培养8d的MtbAT细胞分别加入Mtb—Ag、Rottlerin＋Mtb-Ag、PMA、Rottlerin＋PMA刺激4h，收集细胞用荧光抗体染色后，流式细胞术（FCM）检测CD62L在18T细胞表面的表达。结果体外激活培养6～8d的18T细胞表面CD62L的表达率为75．0％～87．0％。PMA刺激78T细胞3～12h时CD62L表达逐渐下调，表达率为42．3％～25．5％；但在刺激后24h时，又明显回升至53．2％。而PMA＋IMN刺激3、6h时，18T细胞CD62L表达也下调，表达率分别为52．1％、39．3％；但在刺激12h和24h时CD62L表达率分别为52．9％和35．3％。在PMA刺激γδT细胞同时加入Rottlerin，则CD62L的表达率（47．9％）明显高于PMA单独刺激组（31．8％）；MtbAT用Mtb—Ag再刺激4h后，γδT细胞CD62L的表达率由70．0％降为54．8％，而在Mtb．Ag再刺激时加入Rottlerin，则CD62L表达率增加到63．1％。结论γδT细胞在PMA或Mtb-Ag刺激后CD62L的表达下调可被PKCθ特异性抑制剂部分阻断，表明CD62L在γδT细胞表面的表达可能与PKCθ信号转导途径的调控相关。Objective To investigate the role of PKCθ signal pathway on regulation of L-selectin （CD62L） expression in human activated γδT cells. Methods Human peripheral blood mononuclear cells （PBMC） were stimulated with Mycobacterium tuberculosis antigen （Mtb-Ag） and cultured for 6-8 d to generate Mtb-Ag activated T cells（MtbAT） as γδT cells enrichment T cell line. The MtbAT were stimulated with PMA or PMA ＋ IMN for 3, 6, 12 and 24 h, respectively, or MtbAT cultured for 8 d were stimulated with Mtb-Ag, or PMA, with or without PKC0 inhibitor Rotflerin for 4 h. After the treated cells stained with fluorescent labeled monoclonal antibodies, the expression of CD62L on ＂vST cells were measured by flow cytome- try （FCM）. Results The expression of CD62L on γδT cells cultured for 6-8 d were 75.0% -87.0%. Decrease of CD62L from the surface of γδT cells by 3 h to 12 h after exposure to PMA （42.3% to 23.5% ）, but CD62L expression increased to 53.2% when γδT cells were exposed to PMA for 24 h. The expression of CD62L of γδT cells decreased to 52.1% and 39.3% respectively when γδT cells were exposed to PMA ＋ IMN for 3 h and 6 h. After treated with PMA ＋ IMN for 12 h and 24 h, the expression of CD62L were 52.9% and 35. 3% respectively. The CD62L expression of γδT cells treated with PMA and Rottlerin （47.9%） were higher than that treated with PMA alone （31.8%）. After Mtb-Ag restimulated MtbAT for 4 h, the CD62L level of γδT cells decreased from 70.0% to 54.8%, Rottlerin could inhibite Mtb-Ag down regulation CD62L level of γδT cells （63.1%）. Conclusion The CD62L expression of γδT cells could be inhibited partly by the inhibitor of PKCθ, it suggested that PKC0 signal pathway may regulate L-selectin （CD62L） expression of activated human γδT cells.

关 键 词：L-选择素 ΓΔT细胞 蛋白激酶C 

分 类 号：R378[医药卫生—病原生物学]