经卡介苗刺激后结核性胸腔积液中记忆性γδT细胞的细胞因子水平及其受体表达

Cytokine production and clonal analysis of memory γδT cells from patients with tuberculous pleurisy induced by bacille calmette guerin

目的 观察卡介苗刺激后结核性胸膜炎患者的胸腔积液细胞中γδ T细胞的细胞因子及细胞受体表达,了解γδ T细胞在结核病局部细胞免疫应答中的作用.方法 分离结核性胸膜炎患者的胸腔积液细胞,经卡介苗刺激后检测γδ T细胞的细胞因子分泌、细胞亚群、细胞表型及细胞受体表达特征,并提取mRNA,利用3种Vγ和8种Vδ引物,经逆转录PCR扩增cDNA,将PCR产物进行基因扫描分析γδ T细胞各亚家族克隆性.结果 卡介苗刺激胸腔积液细胞后,CD4＋和γδ T细胞分泌γ-干扰素的阳性率分别为0.38%和5.35%,且γδ T细胞分泌γ-干扰素的阳性率远高于CD4＋ T细胞,分泌γ-干扰素的γδ T细胞主要表达白细胞共同抗原（CD45RO）,其阳性率为73.5%.此外,δ2细胞也分泌γ-干扰素（11.1%）和肿瘤坏死因子-α（25.5%）.与未刺激者相比,卡介苗选择性扩增δ2细胞,由多克隆或双克隆转变为寡克隆.结论 卡介苗刺激结核性胸膜炎患者的胸腔积液细胞能诱导记忆性γδT细胞分泌细胞因子,卡介苗体外扩增γδT细胞后,基因扫描显示Vδ2出现寡克隆性.

Objective To evaluate cytokine production and expression of γδ T cells within pleural fluid cells （PFCs） from patients with tuberculous pleurisy following bacille calmette guerin （BCG）stimulation. Methods PFCs were isolated from patients with tuberculous pleurisy, and assessed for cytokine production, cell subpopulation, phenotype and characterization of T cell receptors after stimulation with BCG. The positive PCR products were further labeled with fluorescence and analyzed by genescan technique to determine the CDR3 size and evaluate the clonality of the detectable TCR Vγ and Vδ T cells.Results Following stimulation with BCG, the positivity of interferon-γ（IFN-γ）-producing CD4 T cells and γδ T cells were 0.38% and 5.35%, respectively. Phenotypic analysis indicated that the majority of IFN-γ＋γδ＋ T cells expressed CD45RO ＋ （73.5%）. In addition, δ2 T cells produced IFN-γ （ 11.1% ） and TNF-α（25.5% ）. After expansion with BCG for 3 weeks, cells were harvested and mRNA extracted and RT-PCR conducted to amplify cDNA with 3 primers for Vγ and 8 primers for Vδ. The results indicated that BCG selectively expanded δ2 T cells with oligoclonal peak in Vδ2 cells. Conclusions BCG induced memory γδ and δ2 T cells to produce cytokines in PFCs. Genescan analysis showed that Vδ2 displayed oligoclonality.