CCL19在妊娠期调节性T细胞迁移和调控中的作用机理研究

In unexplained spontaneous abortion during pregnancy, the number of Treg cells was significantly reduced. Treg cells regulate the body's immune system balance and regulate immune tolerance. Where the Treg cells migration and the mechanism of action has not yet been elucidated. CCL19 has a role in regulating Treg migration and DCs can induce differentiation of Treg cells thereby establishing immune tolerance. Our previous study found that CCL19 expression was significantly reduced in spontaneous abortion tissue. CCL19 through its specific binding to the receptor CCR7 regulation of cell function, CCR7 is an important chemokine receptor that induced mature DC migrates to the secondary lymphoid organs. So we propose the hypothesis of CCL19 related to Tregs recruitment and its play immunomodulatory effects of maternal-fetal interface. In order to further investigate the mechanism of CCL19 regulation of Treg, In this study, using multi-color flow cytometry, MACS, two-dimensional electrophoresis time-of-flight mass spectrometry, Western-blot, immunohistochemistry, fluorescence in situ hybridization techniques, and transwell co-culture system, to study the role of CCL19/CCR7 information pathway to the regulatory T cell function in pregnancy, gradually clarify the gestational maternal-fetal immune recognition and tolerance of the molecular mechanism to provide scientific reference for the prevention, diagnosis and treatment of pregnancy-related diseases.

在不明原因自然流产中，Treg细胞数量明显降低。Treg细胞调节机体免疫系统平衡，诱导建立免疫耐受，而Treg的来源、迁移及作用机理至今仍未阐明。CCL19具有调节Treg迁移的作用，DCs能诱导分化产生Treg从而建立免疫耐受。我们前期研究发现CCL19在自然流产组织中表达明显低。CCL19通过与其特异性受体CCR7结合来调节细胞功能，CCR7是诱导成熟DC向次级淋巴器官迁移的重要趋化因子受体。为此，我们提出假说CCL19与母-胎界面Treg的募集、发挥免疫调节作用相关。为了进一步研究其作用机理，本研究采用多色流式细胞术、MACS、二维电泳、飞行时间质谱、Western-blot、免疫组化、FISH和transwell共培养系统等，对CCL19/CCR7在妊娠期对调节性T细胞功能的调控作用进行研究，逐步阐明妊娠母-胎免疫识别和耐受的分子机理，为妊娠相关疾的预防和诊治提供科学参考。

Treg细胞调节机体免疫系统平衡，诱导建立免疫耐受，而Treg的来源、迁移及作用机理至今仍未阐明。CCL19具有调节Treg迁移的作用，DCs能诱导分化产生Treg从而建立免疫耐受。本项目主要研究了人和小鼠自然流产组与对照组母胎界面CCL19与CCR7的表达差异，CCL19在Treg的迁移中的作用，DCs与CCL19的表达对话调节关系，DCs对CCL19表达和细胞间对话的调节功能。通过过继转输调节性T细胞、DC及Transwell共培养技术，流式细胞术检测CCR7及CCL19和Foxp3的表达，研究CCL19对妊娠期调节性T的迁移对妊娠结局的影响。研究结果发现难免流产蜕膜组织中低表达CCR7。小鼠自然流产组Treg细胞表面CCR7的表达显著低于正常对照组，但小鼠蜕膜及胎盘组织中CCR7的表达未有显著差异。自发性流产组小鼠蜕膜组织中CCL19的表达显著低于对照组（p<0.05）。DC与T细胞密切接触能够促进T细胞的分化，进一步刺激阻断培养后，T细胞表达CCL19与对照组相比并未增加。过继转输加入CCL19细胞因子趋化培养的Treg细胞，CBA/J*DBA/2J（对照组CBA/J* DBA/2J仅注射生理盐水），与对照组相比CBA/J*DBA/2J的孕早期7.5天、孕晚期13.5天流产率无统计学差异。这可能与样本数少有关。通过DC与Treg的共培养发现，DC与T细胞密切接触能够促进T细胞的分化，进一步刺激阻断培养后，T细胞表达CCL19与对照组相比并未增加。可能由于DC的迁移导致免疫微环境变化发生转变，蜕膜局部细胞表型Th2 型细胞因子IL-4、粒细胞-巨噬细胞集落刺激因子(GM-CSF)等发生变化。