基于T细胞受体系列检测的脐带血T细胞谱系特点研究
发布时间：2018-10-12 14:23
【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。
[Abstract]:Objective: according to the principle of T cell receptor (TCR) gene rearrangement, the lineage characteristics of (UCB) T cells in umbilical cord blood were analyzed systematically, so as to provide more detailed and comprehensive immunological data for the application of UCB T cells. Methods: (1) the distribution of CD3~, CD4~, CD8~, 伪 尾 T cell and 纬 未 T cell subsets in 16 UCB T cells were detected by flow cytometry. The distribution and clonality of TCR V 伪 / V 尾 subfamilies and UCB TCR V _ 纬 / V 未 subfamilies in 10 cases of UCB and 16 cases of UCB TCR V _ 纬 / V 未 were analyzed by RT-PCR- gene scanning method. The expression levels of three subfamilies of UCB TCR V纬 gene in 16 cases were detected by real-time fluorescence quantitative RT-PCR. (2) T lymphocyte culture combined with PCR- gene scanning method was used. The selective proliferation of TCR V 尾 subfamilies in 3 cases of UCB induced by rhIL-2,PHA,CML cells and PML-RAR 伪 peptide in vitro was analyzed. (3) 12 UCB CD4~ and CD8~ T cells were isolated by MACs method. Then the expression and polymorphism of TCR 味 gene in 60 UCB mononuclear cells and 12 purified UCB CD4~ and CD8~ T cells were analyzed by real-time fluorescence quantitative RT-PCR. Results: (1) the ratios of CD3~ T cells, CD3~ CD8~ T cells, CD3~ TCR 伪 尾 T cells and CD3~ TCR_ 纬 未 T cells in UCB were significantly lower than those in peripheral blood of healthy adults. The ratio of CD3~ TCR 伪 尾 ~ T cells in CD3~ T cells was significantly higher than that in adult peripheral blood, and the percentage of CD3~ TCR_ 纬 未 ~ T cells in CD3~ T cells was significantly lower than that in peripheral blood. (2) the distribution of TCR V 伪 / V 尾 and V _ 纬 / V 未 subfamilies in UCB was highly selective. Cloning analysis showed that V 伪 / V 尾 subfamily in UCB was mainly polyclonal, while V _ 纬 / V 未 appeared clonal proliferative T cells in different subfamilies. This oligoclonal phenomenon was also found in very few V 伪 subfamilies. The quantitative results showed that the distribution of three subfamilies of V _ 纬 gene in UCB was V _ 纬 鈪，
本文编号：2266456
[Abstract]:Objective: according to the principle of T cell receptor (TCR) gene rearrangement, the lineage characteristics of (UCB) T cells in umbilical cord blood were analyzed systematically, so as to provide more detailed and comprehensive immunological data for the application of UCB T cells. Methods: (1) the distribution of CD3~, CD4~, CD8~, 伪 尾 T cell and 纬 未 T cell subsets in 16 UCB T cells were detected by flow cytometry. The distribution and clonality of TCR V 伪 / V 尾 subfamilies and UCB TCR V _ 纬 / V 未 subfamilies in 10 cases of UCB and 16 cases of UCB TCR V _ 纬 / V 未 were analyzed by RT-PCR- gene scanning method. The expression levels of three subfamilies of UCB TCR V纬 gene in 16 cases were detected by real-time fluorescence quantitative RT-PCR. (2) T lymphocyte culture combined with PCR- gene scanning method was used. The selective proliferation of TCR V 尾 subfamilies in 3 cases of UCB induced by rhIL-2,PHA,CML cells and PML-RAR 伪 peptide in vitro was analyzed. (3) 12 UCB CD4~ and CD8~ T cells were isolated by MACs method. Then the expression and polymorphism of TCR 味 gene in 60 UCB mononuclear cells and 12 purified UCB CD4~ and CD8~ T cells were analyzed by real-time fluorescence quantitative RT-PCR. Results: (1) the ratios of CD3~ T cells, CD3~ CD8~ T cells, CD3~ TCR 伪 尾 T cells and CD3~ TCR_ 纬 未 T cells in UCB were significantly lower than those in peripheral blood of healthy adults. The ratio of CD3~ TCR 伪 尾 ~ T cells in CD3~ T cells was significantly higher than that in adult peripheral blood, and the percentage of CD3~ TCR_ 纬 未 ~ T cells in CD3~ T cells was significantly lower than that in peripheral blood. (2) the distribution of TCR V 伪 / V 尾 and V _ 纬 / V 未 subfamilies in UCB was highly selective. Cloning analysis showed that V 伪 / V 尾 subfamily in UCB was mainly polyclonal, while V _ 纬 / V 未 appeared clonal proliferative T cells in different subfamilies. This oligoclonal phenomenon was also found in very few V 伪 subfamilies. The quantitative results showed that the distribution of three subfamilies of V _ 纬 gene in UCB was V _ 纬 鈪，
本文编号：2266456
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