人外周血滤泡辅助T细胞的分选鉴定与功能检测

Isolation and detection of T follicular helper cells in human peripheral blood

目的:探索分选、鉴定和检测人外周血滤泡辅助T细胞(T follicular helper cells,Tfh)功能的方法。方法:使用密度梯度离心(density gradient centrifugation,DGC)、免疫磁珠分选(magnetic-activated cell sorting,MACS)和流式细胞仪(flow cytometry,FCM)分选法来分选人外周血Tfh细胞,分选后使用FCM检测细胞纯度和活性,实时荧光定量PCR检测细胞群的特异性转录因子B细胞淋巴瘤因子(B-cell lymphoma,Bcl)-6 mRNA的表达,激光共聚焦显微镜观察细胞形态和表面分子CD4、CXCR5的表达及定位,鉴定后使用FCM进行功能检测。结果:①FCM检测经分选后Tfh细胞纯度达(87.69±5.01)%,活性达(98.21±0.85)%,纯度和分选前有显著提高(P<0.05)。②实时定量PCR检测分选后的Tfh细胞群的Bcl-6 mRNA相对表达量显著高于分选前(P<0.05),分选后的Tfh细胞群具有高纯度。③激光共聚焦显微镜观察到分选后的Tfh细胞形态完整,CD4及CXCR5的荧光表达于细胞膜并可以融合。④FCM可检测分选鉴定后Tfh细胞功能相关的表面分子PD-1、ICOS、CD40L的表达。结论:采用DGC+MACS+FCM,经鉴定可以得到纯度高、活性好的Tfh细胞,并可以开展其功能检测。

Objective：To explore a method to isolate and detect T follicular helper（Tfh） cells. Methods：Tfh cell in human peripheral blood was isolated by density gradient centrifugation（DGC） plus magnetic-activated cell sorting（MACS） plus flow cytometric sorting （FCS）. Cell purity and cell activity were detected by flow cytometry（FCM）;relative expressions of Bcl6 mRNA by real-time PCR; expressions,location and morphology of cell surface molecules CD4 and CXCR5 by confocal laser scanning microscopy,function by FCM. Results ： ①After sorting, purity of Tfh cell （87.69 ± 5.01 ）%, was significantly enhanced before sorting（P〈0.05） and cell activity was （98.21 ± 0.85）%. ②Expressions of special transcription factor of Tfh cells-Bcl6 were significantly increased after sorting com- pared with those before sorting （P〈0.05） ;after sorting, the purity of the cell was high. ③Morphology of Tfh cell was complete;green fluorescence of CD4 and CXCR5 was expressed in cell membrane and can be fused. ④Expressions of Tfh cell function related cell sur- face molecules PD-1, ICOS and CD40L can be dected by FCM. Conclusions：Tfh cell with high purity and activity can be sorted through ways of density gradient eentrifugation plus MACS plus FCM, then the function of them can be tested by FCM.