TPOAb通过共同抗原决定簇与PD-1/PD-L1途径导致流产的机制研究

The presence of thyroid peroxidase antibodies (TPOAb) in women are associated with abortion, while the pathophysiology of these associations remains largely unknown. Feto-maternal tolerance is necessary for successful pregnancy, however, the imbalance of immune tolerance may result in adverse pregnancy outcomes. Feto-maternal tolerance could be destroyed by complement dependent cytotoxicity (CDC) and PD-1/PD-L1 inhibitory pathway dysfunction. Thus, the mechanisms have been proposed for the association between TPO-Ab and pregnancy loss: first, myeloperoxidase (MPO), which may be the direct targets for TPOAb in endometrium and has the common determinants of TPO, acts with TPOAb in further inducing complement dependent cytotoxicity; Second, PD-1/PDL1 negative costimulatory pathway plays an important role in feto-maternal tolerance. Previously, we had found that the expression of PDL1 decreased in placenta in positive TPOAb murine abortion model, so we infer that TPOAb attenuates the PD1/PDL1 negative coinhibitory pathway and leads to the imbalance of feto-maternal tolerance; so we will explore the possible molecular biology mechanism between TPOAb and miscarriage on the basis of constructing TPOAb positive pregnant mice model from serology, histopathology, protein expression and gene regulation levels. This study will provide theory evidence for screening TPO antibodies during pregnancy and effective immunotherapy to improve pregnancy rate.

母体TPOAb阳性可导致流产风险增加，但机制尚不清楚。母胎耐受是成功妊娠的保障，一旦它被破坏，母体排斥胎儿则导致流产。补体依赖的细胞毒(CDC)与PD-1/PD-L1功能障碍均可破坏母胎耐受。故推测TPOAb破坏母胎耐受的可能机制有：①自身抗体直接的细胞毒作用：TPOAb作用于子宫内膜与TPO有共同抗原决定簇的髓过氧化物酶(MPO)，引起CDC破坏母胎耐受；②PD-1/PD-L1抑制途径介导：该途径是母胎耐受形成过程中至关重要的的负性调节信号，我们的预实验发现TPOAb阳性孕鼠胎盘组织PD-L1表达减少，故推测TPOAb可能使该途径抑制功能减弱，使母胎耐受失衡，导致流产的发生。本项目在已成功构建TPOAb阳性孕鼠模型的基础上，基于上述TPOAb破坏母胎耐受导致流产的科学假说，从血清学、组织病理学、蛋白表达和基因调控等水平探究其分子机制，为妊娠期筛查TPOAb与未来临床干预靶点提供理论依据。

背景：正常甲状腺孕妇的甲状腺自身免疫性疾病与流产有关。研究表明，PD-1/PD-L1信号通路通过促进Treg细胞发育和抑制T辅助物，在母胎耐受中起重要作用。Th17细胞反应，因此对于正常的妊娠维持至关重要。但是，在TAI中尚未完全研究PD-1/PD-L1途径是否导致流产以及具体的机制和原因。.主要研究内容：用甲状腺球蛋白免疫的CBA/J雌性小鼠建立TAI胎儿丢失模型。通过流式细胞仪和PCR检测胎盘和脾脏细胞中Th17,Treg,PD-1和PD-L1的含量。应用免疫组织化学法与Western blot检测胎盘与脾脏中Blimp-1,PD-1,PD-L1,mTOR及Foxp3蛋白的表达。应用RNA干扰（RNA interference,RNAi）技术，阻断人绒毛膜滋养层细胞中的PD-L1蛋白的表达。应用免疫组织荧光法和Western blot检测HTR8细胞中PD-L1,p-ERK,MMP-2,MMP-9的表达水平。.重要结果：与Con组相比，mTg组，CD4+IL-17+T细胞亚群的胎盘和脾脏CD4+CD25+Foxp3+T淋巴细胞数量明显减少（P<0.05）。胎盘中RORγt和IL-17 mRNA的表达升高（P<0.05），胎盘和脾脏中Treg/Th17的比例降低（P<0.05）。用mTg免疫的小鼠胎盘和脾脏中CD4+T的Treg细胞的PD-1和PD-L1的表达减少了。Blimp-1,mTOR在胎盘和脾脏的表达量高于对照组（P<0.05）；Foxp3低于对照组（P<0.05）。敲降PD-L1基因的胚胎滋养细胞PD-L1，磷酸化ERK,MMP-2和MMP-9的表达含量显着降低（P<0.05）。.关键数据：自身免疫性甲状腺炎是因为PD-1/PD-L1含量的降低，一方面影响Treg/Th17比例以及Blimp-1蛋白的含量影响母胎免疫平衡，另一方面使ERK的磷酸化水平减弱影响胚胎滋养层细胞侵袭力而导致流产。.科学意义：本项目对机制的研究将为自身免疫性甲状腺炎患者的治疗提供新的靶点，提高自身免疫性甲状腺炎患者的妊娠成功率。