脐带血和成人外周血TCR Vγ和Vδ亚家族的分布和克隆性研究

Distribution and clonality of TCR Vγ and Vδ subfamily T cells in umbilical cord blood and human peripheral blood

目的探讨脐带血和健康成人外周血TCR Vγ和Vδ基因谱系的分布情况及克隆性特点。方法利用RT-PCR法检测16例脐带血和10例健康成人外周血单个核细胞TCRVγ(Ⅰ～Ⅲ)和Vδ(1～8)各亚家族的表达,了解各亚家族的分布和利用情况;阳性的PCR产物进一步经荧光素标记和基因扫描分析其CDR3长度,了解T细胞的克隆性。2例成人胸腺组织作为对照。结果脐带血T细胞的Vγ基因表达主要集中在VγⅠ和VγⅡ,而Vδ基因则平均表达(4.63±1.03)个亚家族,主要集中在Vδ1、2、3、8中表达。健康成人外周血T细胞除了1例不表达VγⅢ之外,其余均表达全部Vγ基因,而在Vδ基因中则平均表达(3.6±0.52)个亚家族,并以Vδ1、2和3为多见,Vδ4和Vδ5未能在外周血中检测到,而Vδ5和Vδ8的表达则明显低于脐带血(P=0.009,P=0.001);2例成人胸腺组织表达除Vδ4和Vδ6之外的其他Vγ和Vδ亚家族。基因扫描显示16例脐带血中有1例在VγⅠ和5例分别在Vδ2、Vδ4和Vδ6中出现寡克隆性,10例成人外周血中有9例在不同亚家族中出现克隆性T细胞,其余均呈多克隆性。结论脐带血TCRVγ和Vδ亚家族T细胞和健康成人外周血Vδ亚家族T细胞的分布均存在选择性,TCR Vδ亚家族在健康成人外周血中有一定的限制性,其出现克隆性T细胞的频率较脐带血高。

Objective To investigate the distribution and clonality of TCR Vγ and Vδ subfamily T cells in umbilical cord blood and human peripheral blood. Methods The CDR3 of TCR Vγ （ Ⅰ - Ⅲ ） and Vδ （ 1 - 8） subfamily genes in mononuclear cells were amplified from 16 cases of umbilical cord blood （UCB） and 10 cases of healthy adults peripheral blood, to observe the distribution and utilization of TCR Vγ and Vδ repertoire. The positive PCR products were further labeled with fluorescent and analyzed by genescan technique to determine the CDR3 size and evaluate the clonality of the detectable TCR Vγ and Vδ T cells. Thymus tissues of two healthy adults served as controls. Results The Vγ Ⅰ and Vγ Ⅱ were predominantly expressed in UCB. 4.63 ± 1.03 out of 8 Vδ subfamilies were selectively expressed in T cells from UCB, predominantly for Vδ1, 2, 3 and 8. Whereas all Vγ subfamily were expressed in all of peripheral blood from healthy adults except for Vγ Ⅲ, which was detected in 9 out of 10 cases. 3.6 ± 0.52 out of 8 Vδ subfamily T cells were selectively expressed in peripheral blood of healthy adults, predominantly for Vδ1, 2 and 3, while the Vδ4 and Vδ5 were not detected in peripheral blood. Compared with the UCB, a significant lower frequency of Vδ5 and Vδ8 were identified in T cells from healthy adults. All Vγ and Vδ subfamily were expressed in two thymus tissues except for Vδ4 and Vδ6. Genescan analysis showed that the majority of PCR products from UCB displayed multi-peaks except for six cases, which displayed oligoclonal peak in Vγ Ⅰ , Vδ2, Vδ34 and Vδ6, respectively. In contrast, some Vγ and Vδ subfamily products from 9 of 10 cases of healthy adults contained at least an oligoclonal peak in different Vγ and Vδ subfamilies respectively. Conclusion Selected usage could be identified in the TCR Vγ and Vδ subfamily T ceils of UCB and the Vδ subfamily T cells of peripheral blood, respectively. A clear tendency towards restricted usage of Vδ subfamily T cells appears in peripheral blood, and a higher oligoclonality frequency could be found from peripheral blood of healthy adults rather than from UCB.