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目的:探讨经T细胞受体(TCR)途径激活人γδT细胞时,新型蛋白激酶PKCθ在促进转录因子NF-κB活化中的作用。方法:常规分离健康人外周血单个核细胞(PBMC),用结核杆菌耐热性多肽抗原(MtbAg)诱导扩增,获得Vγ9Vδ2 T细胞富集的细胞群(MtbAT)。PKCθ抑制剂(Rottlerin)预处理MtbAT,抗CD3单克隆抗体(mAb)刺激后收集细胞,通过电泳迁移率变动分析(EMSA)检测转录因子NF-κB活性变化;经流式细胞术(FCM)检测T细胞活化分子CD69的表达情况。结果:γδT细胞经抗CD3 mAb刺激,NF-κB活性增强;Rot-tlerin预处理使抗CD3 mAb诱导的NF-κB活化程度显著减弱,并抑制T细胞活化分子CD69的表达。结论:PKCθ在人γδT细胞经TCR途径激活NF-κB过程中具有重要作用。
AIM: To investigate the role of a novel protein kinase PKCθ in activating transcription factor NF-κB when human γδT cells are activated by the T cell receptor (TCR) pathway. Methods: Peripheral blood mononuclear cells (PBMCs) were isolated from healthy volunteers and amplified with MtbAg. The cell population enriched in Vγ9Vδ2 T cells (MtbAT) was obtained. Cells were harvested after stimulation with MtbAT by PKCθ inhibitor (Rottlerin) and stimulation by anti-CD3 monoclonal antibody (mAb). The changes of transcription factor NF-κB activity were detected by electrophoretic mobility shift assay (EMSA). Flow cytometry T cell activation molecule CD69 expression. Results: γδT cells were stimulated with anti-CD3 mAb and the activity of NF-κB was enhanced. Pretreatment with Rot-tlerin attenuated the activation of NF-κB induced by anti-CD3 mAb and inhibited the expression of CD69. Conclusion: PKCθ plays an important role in the activation of NF-κB in human γδT cells via TCR pathway.