【摘要】： 目的:根據(jù)T細(xì)胞受體(TCR)基因重排原理,系統(tǒng)地分析臍帶血(UCB)T細(xì)胞譜系特點等,為UCB T細(xì)胞的應(yīng)用提供更詳細(xì)和更全面的免疫學(xué)特性資料。 方法:(1)利用流式細(xì)胞術(shù)檢測16例UCB T細(xì)胞中CD3~+、CD4~+、CD8~+、αβ~+T細(xì)胞、γδ~+T細(xì)胞各亞群的分布情況,利用RT-PCR-基因掃描法分析10例UCB的TCR Vα/Vβ各亞家族和16例UCB TCR V_γ/Vδ各亞家族的分布和克隆性情況;并利用實時熒光定量RT-PCR檢測16例UCB TCR V_γ基因3個亞家族的表達(dá)水平。(2)采用T淋巴細(xì)胞培養(yǎng)結(jié)合PCR-基因掃描法,分析3例UCB經(jīng)rhIL-2、PHA、CML細(xì)胞和PML-RARα多肽等體外誘導(dǎo)前后其TCR Vβ各亞家族的選擇性增殖情況。(3)采用MACs法分選12例UCB的CD4~+和CD8~+T細(xì)胞,然后利用實時熒光定量RT-PCR分析60例UCB單個核細(xì)胞以及12例純化后UCB CD4~+和CD8~+T細(xì)胞中TCRζ基因的表達(dá)水平及多態(tài)性情況。60例健康成人外周血作為對照。 結(jié)果:(1) UCB中CD3~+T細(xì)胞、CD3~+CD8~+T細(xì)胞、CD3~+TCRαβ~+T細(xì)胞、CD3~+TCR_γδ~+T細(xì)胞在淋巴細(xì)胞中的比值均顯著低于健康成人外周血,而其CD3~+TCRαβ~+T細(xì)胞在CD3~+T細(xì)胞中的比值顯著高于成人外周血,CD3~+TCR_γδ~+T細(xì)胞在CD3~+T細(xì)胞中所占的比例則顯著低于外周血。(2) UCB中TCR Vα/Vβ和V_γ/Vδ亞家族的分布均存在選擇性,克隆性分析顯示UCB中的Vα/Vβ亞家族主要呈多克隆性,而V_γ/Vδ則在不同亞家族中出現(xiàn)克隆性增殖的T細(xì)胞,這種寡克隆現(xiàn)象也見于極個別的Vα亞家族。定量結(jié)果顯示:V_γ基因3個亞家族在UCB中的分布為V_γⅠ＞V_γⅢ＞V_γⅡ,與外周血(V_γⅡ＞V_γⅠ＞V_γⅢ)的分布模式不同。(3)經(jīng)rhIL-2和PHA分別刺激2周后,UCB TCR Vβ亞家族T細(xì)胞表達(dá)全部24個Vβ亞家族,并全部呈多克隆性,而經(jīng)CML細(xì)胞和PML-RARα多肽誘導(dǎo)2周后則分別在Vβ21、Vβ13、Vβ14和Vβ16中呈現(xiàn)克隆性增殖的T細(xì)胞。(4) 60例臍帶血均全部表達(dá)TCRζ基因,相對mRNA表達(dá)量為6.7%±5.56%,而CD4~+和CD8~+T細(xì)胞的TCRζ基因相對mRNA表達(dá)量分別為6.74%±2.0%和6.88%±1.76%,三者的TCRζ基因表達(dá)量均明顯高于健康成人(P=0.000,P=0.034,P=0.000)。所檢測樣本均未發(fā)現(xiàn)國外文獻(xiàn)報道的TCRζ鏈剪接異構(gòu)體ζ-Q。 結(jié)論:本研究首次在國內(nèi)系統(tǒng)全面提供了UCB T細(xì)胞的譜系特點。UCB TCR Vα/Vβ或V_γ/Vδ中存在的不同程度的傾斜性分布和缺失性表達(dá),可能可以作為解釋UCB移植后GVHD發(fā)生率較低的部分原因;而UCB中存在的克隆性T細(xì)胞則提示可能與T細(xì)胞發(fā)育過程中出現(xiàn)的克隆發(fā)育不全有關(guān)。UCB T細(xì)胞在體外rhIL-2和PHA等細(xì)胞因子刺激下,具有表達(dá)全部Vβ亞家族的潛能,而在CML細(xì)胞和PML-RARα多肽等白血病相關(guān)抗原誘導(dǎo)下,則具有限制性和克隆性增殖的能力。此外,本研究還率先建立了檢測TCR V_γ基因和TCRζ基因的實時熒光定量RT-PCR比較Ct法,并首次提供這兩個基因在UCB中的表達(dá)情況。
[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~, 偽 尾 T cell and 緯 未 T cell subsets in 16 UCB T cells were detected by flow cytometry. The distribution and clonality of TCR V 偽 / V 尾 subfamilies and UCB TCR V _ 緯 / V 未 subfamilies in 10 cases of UCB and 16 cases of UCB TCR V _ 緯 / V 未 were analyzed by RT-PCR- gene scanning method. The expression levels of three subfamilies of UCB TCR V緯 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 偽 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 偽 尾 T cells and CD3~ TCR_ 緯 未 T cells in UCB were significantly lower than those in peripheral blood of healthy adults. The ratio of CD3~ TCR 偽 尾 ~ T cells in CD3~ T cells was significantly higher than that in adult peripheral blood, and the percentage of CD3~ TCR_ 緯 未 ~ T cells in CD3~ T cells was significantly lower than that in peripheral blood. (2) the distribution of TCR V 偽 / V 尾 and V _ 緯 / V 未 subfamilies in UCB was highly selective. Cloning analysis showed that V 偽 / V 尾 subfamily in UCB was mainly polyclonal, while V _ 緯 / V 未 appeared clonal proliferative T cells in different subfamilies. This oligoclonal phenomenon was also found in very few V 偽 subfamilies. The quantitative results showed that the distribution of three subfamilies of V _ 緯 gene in UCB was V _ 緯 鈪，

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