蛋白激酶Cθ在γδT细胞表达L-选择素中的调控作用

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)中的凋控作用.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6～8 d,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、24 h,或用培养8 d的MtbAT细胞分别加入Mtb-Ag、Rot-tlerin+Mtb-Ag、PMA、Rottlerin+PMA刺激4 h,收集细胞用荧光抗体染色后,流式细胞术(FCM)检测CD62L在γδT细胞表而的表达.结果 体外激活培养6～8 d的γδT细胞表面CD62L的表达率为75.0%～87.0%.PMA刺激γδT细胞3～12 h时CD62L表达逐渐下调,表达率为42.3%～23.5%;但在刺激后24 h时,又明显回升至53.2%.而PMA+IMN刺激3、6 h时,γδT细胞CD62L表达也下调,表达率分别为52.1%、39.3%;但在刺激12 h和24 h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4 h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火.%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 Rottlerin for 4 h. After the treated cells stained with fluo-rescent labeled monoclonal antibodies, the expression of CD62L on γδT 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 ingibited partly by the inhibitor of PKCθ signal pathway may regulate L-selectin (CD62L) expression of activated human γδT cells.