过继转输T调节细胞对弓形虫感染致不良妊娠结局影响的分子机制研究

During human pregnancy, Toxoplasma gondii infection can cause unfavorable pregnancy outcomes through vertical transmission.It have been approved that decidual T regulatory(Treg) cells play an important role on sustain the normal pregnancy.In the previous study we have found that the change of the Treg's amount and functions contributed to abnormal outcome with Toxoplasma gondii infection during pregnancy.In this study the Treg cells were purifed from GFP-Foxp3 Transgenic mice, so the Treg cells were show an green flurescent because the recombinant GFP with Foxp3 protein expressed.The green flurescent Treg cells purifed from normal mice and Toxoplasma gondii infected mice respectively.The above two kinds of green fluresenct Treg cells will transfered into the Toxoplasma gondii infected BABL/c pregnant mice respectively to value their therapy effect on the abnormal pregnant outcome.The distribution and the amount of exotic Treg cells in the BABL/c mice were observed through vivi animal imaging system.The functional moleculares(CTLA-4,TGF- Foxp3) of Treg were analyzed with the methods fo moleculare biology after the therapy of transferred Treg cells. Also the relationship between the effect of transferred Treg cells and the abnormal pregnant outcome were analyzed.We expect this work may shed light on the molecular mechanism of the effect of transferred Treg on the abnormal pregnant outcome induced by Toxoplasma gondii infection during pregnancy.The exotic Treg cells will be provided a new therapeutic target for the treatment on adverse pregnancy outcomes of pregnant mice infected by T. gondii.The result of this study might be considered as potentially useful therapy to improve the abnormal pregnant outcome by Toxoplsma gondii infection during pregnancy.

孕期弓形虫感染可垂直传播造成不良妊娠结局的发生。已证实蜕膜Treg细胞对于维持正常妊娠至关重要，申请人已研究发现，孕期弓形虫感染致不良妊娠结局的发生与孕鼠脾脏和胎盘Treg细胞数量及功能改变密切相关。本研究拟采用绿色荧光蛋白转基因（GFP-Foxp3 TG，Foxp3+Treg细胞呈自发绿色荧光）小鼠，将正常妊娠及早孕期弓形虫感染鼠Treg过继转输早孕期弓形虫感染鼠，观察对不良妊娠结局的影响，并采用活体成像系统观察外源性Treg被募集到蜕膜的数量及其与妊娠结局改善程度的关系，同时采用分子生物学方法检测Treg功能相关分子及治疗前后母胎界面各主要分子及细胞因子的表达水平。旨在探索过继转输Treg细胞对弓形虫感染致不良妊娠结局影响的分子机制。迄今未见相关报道。本研究组一直致力于弓形虫感染致不良妊娠结局发生机制的研究，本研究将为寻找减少弓形虫感染后不良妊娠结局发生的有效手段和方法奠定理论基础。

正常妊娠的维持有赖于母胎界面免疫微环境的稳定，打破这种微环境就可能导致不良妊娠结局的发生。蜕膜Treg细胞（regulatory T cells，Treg）是构成母胎界面免疫微环境的重要细胞，本室研究已发现，孕期弓形虫感染将导致蜕膜Treg细胞数量及其负性调节功能的改变；然而，Treg细胞在孕期弓形虫感染所致不良妊娠结局发生中的确切分子机制，以及过继转输蜕膜Treg细胞能否有效改善不良妊娠结局仍待深入研究。.本项目通过建立弓形虫感染孕鼠模型，深入探讨了蜕膜Treg细胞及其相关分子在弓形虫感染致不良妊娠结局中的作用机制，进而观察了过继转输Treg细胞对弓形虫感染所致不良妊娠结局的影响及其机制；本课题还探索了蜕膜Treg细胞所分泌的主要细胞因子IL-10在改善早孕期弓形虫感染致不良妊娠结局中的机制。此外，为了筛选弓形虫感染致不良妊娠结局发生的分子靶点，采用无标记液相色谱-串联质谱方法对弓形虫感染所致胎盘差异蛋白进行了筛选。主要研究结果如下：.⑴ 弓形虫感染导致蜕膜Treg细胞CTLA-4和PD-1表达水平降低，促进Treg细胞凋亡，数量减少，Treg细胞负性调节功能降低；还导致蜕膜Treg细胞细胞因子TGF-β、IL-10分泌减少，这打破了母胎界面维持正常妊娠所需要的免疫微环境，从而导致不良妊娠结局的发生。.⑵ 过继转输正常妊娠蜕膜Treg细胞可增加受体孕鼠体内蜕膜Treg细胞表面CTLA-4和PD-1的表达，从而增加受体孕鼠体内的Treg细胞的免疫抑制功能；此外，过继转输蜕膜Treg细胞可以纠正弓形虫感染所致母胎界面细胞因子的失衡，进而改善弓形虫感染所致不良妊娠结局。.⑶ 孕期弓形虫感染可促进蜕膜Treg细胞和滋养层细胞的凋亡；IL-10治疗可以调控凋亡信号分子Caspase-3和Caspase-8的表达，从而有效减少弓形虫感染所致蜕膜Treg细胞和滋养层细胞的凋亡。.⑷ 通过无标记液相色谱-串联质谱技术，从792种检测的胎盘蛋白中筛选出58种在弓形虫感染前后有差异表达的蛋白，这些蛋白多与滋养层细胞侵袭、胎盘发育、宿主免疫应答等生命过程密切相关。.以上研究结果有望在寻找改善弓形虫感染所致不良妊娠结局的新的免疫方法和策略方面获得突破。