sHLA-G在孕期弓形虫感染致不良妊娠结局中的作用机制研究

The incidence of adverse pregnancy outcome caused by the disturbance of maternal-fetal immune tolerance is the most harmful result of Toxoplasma gondii infection. Lots of study demonstrated that the fetal extravillous trophocytes, especially the soluble human leukocyte antigen -G (sHLA-G) secreted by these cells played a key role in the establishment and maintenance of the maternal-fetal immune privileged site..Hereby, we intended to infect pregnant C57BL/6 mice with Toxoplasma gondii RH strain. 0, 2, 4 and 6 days after infection, we will detect and compare the expression level of sHLA-G in the peripheral blood of the infected and non-infected mice. According to these data, we will kill the mice at the optimal time after Toxoplasma gondii infection and detect the mRNA and protein level of sHLA-G in the placental tissues. Also, the pregnancy outcome should be recorded. Using statistical software, we could analyze the correlation between the expression level of sHLA-G and the pregnancy outcome in Toxoplasma gondii infected pregnant mice. To further reveal the mechanism, we will infect the first trimester human trophocytes with Toxoplasma gondii RH strain. To get the optimal infection dose and time, the expression level of sHLA-G in the supernant should be detected repeatedly. Next, we will add the supernatant into the culture media of natural killer cells (NK cells) and mixed lymphocytes respectively. The expression of inhibitory receptors on NK cells, the cytotoxic function of NK cells, the number and supression function of regulatory T cells (Treg) cells and the expression of FoxP3 and TGF-β on Treg cells will be detected in sequences. Then, recombinant sHLA-G or anti- sHLA-G antibody into the supernatant will be added before their addition into the culture media. We will repeat the above detection to confirm the effects of sHLA-G on NK cells and Treg cells..We aim to demonstrate the effects and mechanism of sHLA-G on the incidence of adverse pregnancy outcome during Toxoplasma gondii infection and search the method to reduce the neonatal birth defects. Until now, there has been no report about this aspect.

弓形虫感染的最大危害是孕期感染导致母胎免疫耐受失衡从而引发不良妊娠结局。大量研究表明，胎儿绒毛膜滋养层细胞分泌的sHLA-G在建立与维持母胎界面免疫微环境中发挥重要作用。本研究拟制备弓形虫强毒株（RH株），建立感染动物模型，检测孕期弓形虫感染后sHLA-G表达水平的变化及其与妊娠结局的关系；为了进一步探讨其作用机制，分离人工流产的早孕期滋养层细胞，弓形虫体外感染后将培养上清添加至NK细胞和混合淋巴细胞培养体系中，检测NK细胞表面免疫抑制性受体的表达水平及其细胞毒效应，Treg细胞数量、相关分子表达水平及其抑制功能；然后在培养上清中添加外源性重组sHLA-G或采用anti-sHLA-G抗体阻断的方法来确证sHLA-G对NK细胞和Treg细胞的作用。研究成果将进一步揭示sHLA-G在孕期弓形虫感染致不良妊娠结局中的作用机制，为寻找减少弓形虫感染后不良妊娠结局发生的有效方法奠定理论基础。

大量研究表明，胎儿绒毛膜滋养层细胞分泌的sHLA-G可与母体蜕膜中含量最高的NK细胞表面的杀伤抑制性受体结合，从而抑制母体蜕膜NK 细胞对胎儿的杀伤排斥，这是母胎界面的免疫系统维持正常妊娠的保障之一。另有研究证实，sHLA-G可通过Fas-FasL途径促进NK细胞的溶解。然而，孕期弓形虫感染对母胎界面sHLA-G表达的影响目前众说纷纭，sHLA-G 与蜕膜NK细胞的相互作用是否参与了弓形虫感染致不良妊娠结局的发生至今仍不清楚。.本研究在课题组前期研究的基础上，探讨了孕期弓形虫感染对母胎界面sHLA-G表达的影响以及sHLA-G在弓形虫感染导致的蜕膜NK细胞凋亡中的机制研究，取得研究结果如下：.①弓形虫感染可上调滋养层细胞sHLA-G的分泌水平：弓形虫体内感染可致孕鼠外周血、羊水及胎盘组织上清中Qa-2的浓度升高，相关性分析结果表明，感染组孕鼠的胚胎吸收率与其Qa-2的表达水平正相关；弓形虫体外感染人滋养层细胞后，其上清sHLA-G的浓度显著升高，且随着感染时间的延长，其水平逐渐升高。.②sHLA-G参与了弓形虫感染导致的蜕膜NK细胞凋亡：自早孕期自愿流产者的蜕膜和绒毛组织分离纯化得到人蜕膜NK细胞和人滋养层细胞，成功建立了弓形虫感染的NK细胞与滋养层细胞的共培养体系；弓形虫感染可上调共培养体系中sHLA-G的水平、蜕膜NK细胞的凋亡及其caspase3和 caspase8的表达；利用sHLA-G中和抗体证实了sHLA-G参与蜕膜NK细胞的凋亡过程。.本研究表明，弓形虫感染可上调滋养层细胞sHLA-G的水平，高表达的sHLA-G可进一步促进蜕膜NK细胞的凋亡。该结果揭示了弓形虫感染致不良妊娠结局的一重要免疫机制，对减少出生缺陷，提高出生人口质量有理论借鉴价值。