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

摘要:

目的：比较全外显子组数据分析中不同的预处理方法和变异过滤方法对变异识别的影响。方法：利用２例全外显子组测序数据，从使用不同的预处理方法（FASTX-Toolkit、Trimmomatic及未做预处理）、修饰后不成对读长（single-end reads，SE）取舍策略以及变异过滤方法[Hard过滤和变异质量得分重新校正（variant quality score recalibration，VQSR）]3个方面，通过数据覆盖深度（depth of coverage，DP）、识别变异的数目、转换/颠换比值和基因型一致性等特征，比较他们对全外显子组变异识别结果的影响。结果：Trimmomatic预处理后的读长测序ＤP与未预处理的原始数据接近，但明显高于FASTX-Toolkit预处理方法。当DP≥10×且基因型质量分数（genotype quality score，GQ）≥20时，经Trimmomatic预处理后识别到的单核苷酸变异（single nucleotide variant，SNV）数量比FASTX-Toolkit多，与未预处理组接近。当包含SE时，FASTX-Toolkit组多识别出的SNV数量高于（28%）Trimmomatic组（5%）。当样本量较少时，在所有实验组中Hard过滤方法滤掉的SNV要少于VQSR。结论：Trimmo－matic修饰（过滤）原始序列更温和，而FASTX-Toolkit可能过度过滤了原始数据。保留SE有利于下游变异识别。Hard过滤相较于VQSR表现出了更高的容忍度。

Abstract:

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 centrifugation plus MACS plus FCM，then the function of them can be tested by FCM.