免疫组库测序分析新生儿脓毒症患者外周血T细胞受体β链CDR3的多样性

Diversity of the T cell receptor β chain complementarity-determining region 3 in peripheral blood of neonates with sepsis:an analysis based on immune repertoire sequencing

目的采用免疫组库测序的方法分析新生儿脓毒症患者外周血T细胞受体(T cell receptor,TCR)β链互补决定区3 (complementarity determining region 3,CDR3)的多样性,探讨新生儿脓毒症的可能发病机制。方法 12例新生儿脓毒症确诊患儿为病例组,9例胎龄、出生体重及日龄相匹配的健康足月儿为对照组。采用Omega公司核酸纯化试剂盒(SQ Blood DNA Kit Ⅱ)从外周血样品中提取DNA,对TCRβ链CDR3进行多重PCR扩增,然后对产物进行高通量测序,分析其TCRβ链CDR3多样性及表达的差异。结果病例组和对照组的TCRβ链CDR3长度和类型相似,且均呈高斯分布。采用D50和香农-威纳指数作为多样性的评价指标,显示病例组TCRβ链CDR3多样性低于对照组,差异有统计学意义(P<0.05)。针对TCRβ链V片段48个基因的使用频率进行比较,其中TRBV10-3、TRBV2和TRBV20-1的使用频率病例组高于对照组,差异有统计学意义(P<0.05);对TCRβ链J片段13个基因的使用频率进行比较,其中TRBJ2-3、TRBJ2-5和TRBJ2-7的使用频率病例组高于对照组,差异有统计学意义(P<0.05)。结论新生儿脓毒症患者外周血TCRβ链CDR3多样性发生了明显改变,提示这可能与新生儿脓毒症的免疫发病机制有关。

Objective To investigate the diversity of peripheral blood T cell receptor(TCR) β chain complementarity-determining region 3(CDR3) based on immune repertoire sequencing in neonates with sepsis and the possible pathogenesis of neonatal sepsis. Methods A total of 12 neonates with sepsis were enrolled as the case group,and 9 healthy full-term infants, matched for gestational age, birth weight, and age, were enrolled as the control group.Omega nucleic acid purification kit(SQ blood DNA Kit Ⅱ) was used to extract DNA from peripheral blood samples,TCR β chain CDR3 was amplified by multiplex PCR, and then high-throughput sequencing was performed for the products to analyze the diversity of TCR β chain CDR3 and the difference in expression. Results The length and type of TCR β chain CDR3 were similar between the case and control groups, and Gaussian distribution was observed in both groups. With D50 and Shannon-Wiener index as the evaluation indices for diversity, the case group had a significantly lower diversity of TCR β chain CDR3 than the control group(P<0.05). The frequency of 48 genes in TCR β chain V segment was compared, and the results showed that compared with the control group, the case group had significantly higher frequencies of TRBV10-3, TRBV2, and TRBV20-1(P<0.05). The frequency of 13 genes in TCR β chain J segment were compared, and the results showed that compared with the control group, the case group had significantly higher frequencies of TRBJ2-3, TRBJ2-5, and TRBJ2-7(P<0.05). Conclusions There is a significant change in the diversity of TCR β chain CDR3 in the peripheral blood of neonates with sepsis, suggesting that it might be associated with the immune pathogenesis of neonatal sepsis.