自身免疫甲状腺炎母体流产风险增加与α烯醇化酶抗原的致病作用研究

Autoimmune thyroiditis (AIT) is a very common autoimmune disease. Much evidence has demonstrated a significant increase in the risk of miscarriage among the pregnant women with AIT, which has severely adverse effects on their physical and psychological health. Its pathogenesis is still unclear; however, autoimmune attack on ovary, uterus and placenta may play very important roles, since levothyroxine treatment can only reduce the incidence of miscarriage by 52%. α-Enolase (ENO1) is a multifunctional protein, and mainly expressed on the surface of several cell types including vascular endothelium as plasminogen receptor and in the plasma as a highly conserved key glycolytic enzyme from archaebacteria to mammals. It can be predicted that ENO1-specific autoantibody（ENO1Ab）may have the vascular endothelium injured through inducing the formation of immune complexes, and perturb the intravascular and pericellular fibrinolytic system by interfering the binding of ENO1 with plasminogen. Furthermore, two studies have found the alterations in the expression of ENO1 antigen in the placenta and decidual tissues, which was associated with unexplained miscarriage. Moreover, we have recently found the production of ENO1Ab at different extents in AIT patients and its experimental animal model. All these highly suggest that the autoimmune responses mediated by ENO1 antigen may be the important pathogenic factors which contribute to the increased occurrences of miscarriage in AIT patients. On the basis of our established cohort of AIT pregnant women and ENO1-immunized CBA/J mouse model, we are going to perform a series of epidemiological and experimental studies on the association between the production of ENO1Ab and the occurrence of miscarriage, its potential immune mechanisms at cellular and molecular levels, the specific antigenic epitopes of ENO1 protein related to miscarriage and possible interventions derived from the findings above. The results of this project would greatly contribute to developing the new strategies for the risk prediction, treatment and prophylysis of miscarriage in child-bearing women with AIT.

大量研究证实自身免疫甲状腺炎（AIT）孕妇流产风险显著增加。其危害重，机制不清，补充左甲状腺素仅能减少52%，提示自身免疫因素在流产发生中起重要作用。α烯醇化酶（ENO1）是表达于血管内皮等细胞上的多功能蛋白。理论上ENO1抗体（ENO1Ab）可通过形成免疫复合物损伤血管内皮和干扰其作为纤溶解酶原受体参与的纤溶过程而促进流产发生。现仅有二项相关研究：在不明原因流产的胎盘上ENO1抗原表达降低，蜕膜中升高，但无机制探讨。我们最近发现AIT患者和动物模型中有不同程度ENO1Ab产生。这高度提示ENO1特异性自身免疫可能是导致AIT孕妇流产的重要因素。本项目拟基于已建立的AIT孕妇队列和ENO1免疫鼠模型，利用流调、质谱分析、生物信息技术和荧光染色等手段，研究ENO1Ab与流产的相关性、其致流产的细胞与分子免疫机制、相关ENO1抗原表位和可衍生的预测和干预手段，为AIT孕妇防治流产提供新策略。

自身免疫甲状腺炎（AIT）孕妇流产风险显著增加，危害重，机制不清，自身免疫因素不容忽视。α烯醇化酶（ENO1）在甲状腺和胎盘等处均表达。本项目对甲功正常孕妇研究显示，有自然流产的AIT和非AIT妇女血ENO1特异抗体（ENO1Ab）水平均明显高于相应无流产者，调整其它因素后，血高ENO1Ab水平仍是这些妇女自然流产的独立危险因素。利用生信分析预测出18种可能的ENO1线性抗原表位，命名为P1-P18。对血各表位特异自身抗体的检测表明，P6(aa168-183)和P9(aa252-270)可能是流产相关致病表位。进一步队列研究和分析表明，血ENO1-P6特异总IgG和IgG1、IgG2及IgG3均为甲功正常AIT孕妇自然流产的独立危险因素。ENO1-P9Ab即是甲功正常AIT又是非AIT妇女自然流产发生的独立危险因素。在胚胎植入数≥5的甲状腺球蛋白免疫诱导的实验性自身免疫甲状腺炎（EAT）母鼠中，有流产者血ENO1-P6Ab、ENO1-P6-aa168-175Ab水平显著高于无流产者；血清ENO1-P6Ab表达阳性者较阴性者胚胎吸收率明显增加。提示，血ENO1-P6Ab高表达（尤其ENO1-P6-aa168-175Ab高表达）是促进AIT相关自然流产的重要因素，且此抗体的产生可能继发于甲状腺自身免疫本身，不是由于并存的其它自身免疫病诱导所致。因此，血ENO1-P6Ab测定可作为监测AIT妇女自然流产风险的重要血清标志物。接受ENO1-P6免疫的普通和EAT母鼠的胚胎吸收率均显著高于单纯KLH注射的对照组;但有趣的是，给予ENO1-P9免疫母鼠的胚胎植入数和吸收率无明显变化。相关机制研究发现，在孕早期针对ENO1-P6和P9的自身免疫反应并不通过引起甲状腺本身的自身免疫损害和功能变化而致流产。ENO1-P9免疫母鼠的胎盘局部可能因纤溶酶-抗纤溶酶复合物含量降低而出现高凝状态，后者可能为促进流产发生的一个辅助机制。而补体介导的细胞毒作用是胎盘局部（尤其是滋养层上皮细胞）发生ENO1-P6相关自身免疫损伤的主要机制，最终可致流产发生，但该表位抗体的特异性直接影响尚有待明确。另外，孕前静注ENO1-P6表位特异小肽aa168-175被发现可降低接受ENO1-P6免疫的EAT母鼠的胚胎吸收率。提示，该方法可成为临床上降低甲功正常AIT母体自然流产风险的一个新手段。