【作者】 陈少华；

【导师】 李扬秋；

【作者基本信息】 暨南大学 ， 内科血液病学， 2009， 硕士

【摘要】 目的:根据T细胞受体(TCR)基因重排原理,系统地分析脐带血(UCB)T细胞谱系特点等,为UCB T细胞的应用提供更详细和更全面的免疫学特性资料。方法:(1)利用流式细胞术检测16例UCB T细胞中CD3~+、CD4~+、CD8~+、αβ~+T细胞、γδ~+T细胞各亚群的分布情况,利用RT-PCR-基因扫描法分析10例UCB的TCR Vα/Vβ各亚家族和16例UCB TCR V_γ/Vδ各亚家族的分布和克隆性情况;并利用实时荧光定量RT-PCR检测16例UCB TCR V_γ基因3个亚家族的表达水平。(2)采用T淋巴细胞培养结合PCR-基因扫描法,分析3例UCB经rhIL-2、PHA、CML细胞和PML-RARα多肽等体外诱导前后其TCR Vβ各亚家族的选择性增殖情况。(3)采用MACs法分选12例UCB的CD4~+和CD8~+T细胞,然后利用实时荧光定量RT-PCR分析60例UCB单个核细胞以及12例纯化后UCB CD4~+和CD8~+T细胞中TCRζ基因的表达水平及多态性情况。60例健康成人外周血作为对照。结果:(1) UCB中CD3~+T细胞、CD3~+CD8~+T细胞、CD3~+TCRαβ~+T细胞、CD3~+TCR_γδ~+T细胞在淋巴细胞中的比值均显著低于健康成人外周血,而其CD3~+TCRαβ~+T细胞在CD3~+T细胞中的比值显著高于成人外周血,CD3~+TCR_γδ~+T细胞在CD3~+T细胞中所占的比例则显著低于外周血。(2) UCB中TCR Vα/Vβ和V_γ/Vδ亚家族的分布均存在选择性,克隆性分析显示UCB中的Vα/Vβ亚家族主要呈多克隆性,而V_γ/Vδ则在不同亚家族中出现克隆性增殖的T细胞,这种寡克隆现象也见于极个别的Vα亚家族。定量结果显示:V_γ基因3个亚家族在UCB中的分布为V_γⅠ＞V_γⅢ＞V_γⅡ,与外周血(V_γⅡ＞V_γⅠ＞V_γⅢ)的分布模式不同。(3)经rhIL-2和PHA分别刺激2周后,UCB TCR Vβ亚家族T细胞表达全部24个Vβ亚家族,并全部呈多克隆性,而经CML细胞和PML-RARα多肽诱导2周后则分别在Vβ21、Vβ13、Vβ14和Vβ16中呈现克隆性增殖的T细胞。(4) 60例脐带血均全部表达TCRζ基因,相对mRNA表达量为6.7%±5.56%,而CD4~+和CD8~+T细胞的TCRζ基因相对mRNA表达量分别为6.74%±2.0%和6.88%±1.76%,三者的TCRζ基因表达量均明显高于健康成人(P=0.000,P=0.034,P=0.000)。所检测样本均未发现国外文献报道的TCRζ链剪接异构体ζ-Q。结论:本研究首次在国内系统全面提供了UCB T细胞的谱系特点。UCB TCR Vα/Vβ或V_γ/Vδ中存在的不同程度的倾斜性分布和缺失性表达,可能可以作为解释UCB移植后GVHD发生率较低的部分原因;而UCB中存在的克隆性T细胞则提示可能与T细胞发育过程中出现的克隆发育不全有关。UCB T细胞在体外rhIL-2和PHA等细胞因子刺激下,具有表达全部Vβ亚家族的潜能,而在CML细胞和PML-RARα多肽等白血病相关抗原诱导下,则具有限制性和克隆性增殖的能力。此外,本研究还率先建立了检测TCR V_γ基因和TCRζ基因的实时荧光定量RT-PCR比较Ct法,并首次提供这两个基因在UCB中的表达情况。更多还原

【Abstract】 Objective:To analyze the characteristics of umbilical cord blood(UCB) T cell repertoire based on the detection of T-cell receptor(TCR) gene rearrangement.This study is expected to provide more detailed and more comprehensive information on immunological characteristics of UCB T cells for its application.Method:The frequency of CD3~+-,CD4~+-,CD8~+-,αβ~+- andγδ~+- T cells were examined in umbilical cord blood(UCB) by FACS.The CDR3 size of TCR Vα/Vβand TCR V_γ/Vδsubfamily genes were analyzed in mononuclear cells(MCs) from 10 to 16 UCB samples respectively,using RT-PCR and genescan technique.Quantitative detection of the expression level of TCR V_γsubfamily genes were performed from 16 UCB cases by real-time PCR.T cells from 3 cases UCB were stimulated and amplified by rhIL-2,PHA,CML cell or PML-RARαpeptide in vitro using liquid T-cell culture technique,to detect the usage and clonality feature of TCR Vβsubfamilies.CD4~+-and CD8~+-T cells were sorted from MCs of 12 UCB samples using the MACS magnetic bead separation system.Real-time PCR with SYBR Green I technique was used for detecting TCRζgene expression level in MCs,sorted CD4~+- and CD8~+-T cells from UCB.The mutation or polymorpheirm of TCRζgene was determined on the change of melting curve and nucleotide sequencing.Peripheral blood(PB) samples from total of 60 healthy adult individuals served as controls.Result:(1)The mean percentages of CD3~+/MCs,CD3~+CD8~+/MCs,CD3~+ TCRαβ~+/MCs, CD3~+TCR_γδ~+/MCs and CD3~+TCR_γδ~+/CD3~+ cells from UCB were significantly lower than that from healthy adult individuals,while the CD3~+TCRαβ~+/CD3~+ cells was higer in UCB than that from healthy adult individual PB group.(2) The selected usage of TCR repertoire could be identified in TCR Vα/Vβand TCR V_γ/Vδof UCB.The majority of TCR Vα/Vβsubfamily T cells in UCB displayed polyclonality,whereas the clonal expanded T cells were detected in different TCR V_γ/Vδsubfamilies,occasional oligoclonal peaks are identified in some TCR Vαsubfamilies.The pattern of TCR V_γ,subfamily expression level in UCB was V_γⅠ＞V_γⅢ＞V_γⅡ,in contrast,V_γⅡ＞V_γⅠ＞V_γⅢwas found in PBMCs.(3) The expression and polyclonality pattern of all 24 TCR Vβsubfamily T cells could be detected after stimulation with rhIL-2 or PHA respectively,whereas the restricted expression of TCR Vβsubfamilies and Vβ21,Vβ13,Vβ14 and Vβ16 clonal expansion T cells were found in cultured T cells induced by CML cells or PML-RARαpeptide respectively.(4) TCRζgene was expressed in all cases from both UCB and healthy adults.The relative mRNA expression level of TCRζgene in mononuclear cells(6.7%±5.56%),CD4~+T cells(6.74%±2.0%) and CD8~+T cells (6.88%±1.76%) from UCB were significantly higher than that from healthy adults (P=0.000,P=0.034,P=0.000) respectively.Neither mutation nor polymorpherim of TCRζgene was identfied in all samples.Conclusion:It is the first report providing a detailed profile of the UCB T-cell repertoire in China.The skewed distribution of Vα/Vβor V_γ/Vδsubfamily T cells could be found in UCB,which might be responsible for lower incidence of GVHD in cord blood hematopoietic stem cells transplantation.The clonal expansion of TCR subfamilies in UCB T cells remains unknown,it might be due to clonal absence during T-cell repertoire development.However,UCB T cells may have the potential capability of proliferation in different TCR Vβsubfamily T cells,and the ability of restricted usage and clonal expansion,when T cells were induced by leukemia associated antigen such as CML cells or PML-RARαpeptide.In addition,the SYBR Green I real-time technique for quantitative detection of TCR V_γand TCRζchain expression levels was established successfully.This is,to our knowledge,the first description in the expression feature of TCR V_γand TCRζchain gene in UCB.更多还原