绵羊内源性逆转录病毒调节胚胎滋养层细胞融合发育的机制

Previous research confirmed that the expression of endogenous Jaagsiekte retrovirus (enJSRV) and its receptor Hyaluronidase-2 (Hyal-2) in placenta and conceptus is closely related to the pregnancy and development process. the formation of the placenta is a dynamic physiological process highly regulated by,and trophoblast cells differentiation is the key to placentogenesis. On the basis of these earlier studies, the project proposed is designed to establish 293T cell line stably expressing the enJSRV envelope protein (Env) and Hyal-2 .Then co-culture them and observe the fusion of subsequent cells. Also we explore RNA interference aiming to induce enJSRV-env gene silencing, observe the growth of the primarily cultured trophoblast cells by transfected with the best RNAi plasmid and evaluate the regulatory role of MAPK signal transduction pathway in trophoblast cells differentiation by the technique of immune-precipitation. Package eukaryotic expression vector and RNAi plasmid into the recombinant lentivirus to infect the pregnancy BALB/c mice and observe the pregnancy process and embryo development so as to confirm the in vitro experimental results further which investigate the regulation role of enJSRV in embryo development , especially in trophoblast cells fusion and differentiation process . The research results will provide deep insight into the research of animal survival, development, reproduction and other aspects.

前期的研究证实绵羊内源性逆转录病毒（enJSRV）及其受体HYAL2在不同妊娠期的胎盘和孕体中的表达变化规律均与绵羊的妊娠过程和孕体的发育密切相关。胎盘的发生是一个受到高度调控的动态生理过程，关键环节是滋养层细胞的分化。本项目的提出是在原有研究的基础上通过构建稳定表达enJSRV的Env蛋白及其受体HYAL2的293T细胞系，然后进行共培养观察细胞融合机制并通过RNA干扰诱导enJSRV-env基因沉默，构建并筛选最佳干扰质粒转染原代培养的滋养层细胞，检测细胞生长发育情况，采用免疫沉淀法检测滋养层细胞分化发育中MAPK信号传导通路的调节。同时将构建的超表达质粒和干扰质粒包装慢病毒感染妊娠小鼠，观察小鼠妊娠过程和胚胎发育情况，进一步验证体外细胞中的实验结果，从而揭示enJSRV在胚胎发育特别是滋养层细胞融合分化过程的调控机制。研究成果将对动物的生存、发育、繁殖等具有重要的应用价值和理论意义。

该项目严格按照计划任务书执行，完成了预期的研究目标。已发表了相关学术论文25篇，其中SCI收录4篇和中文核心期刊7篇，完成发布1项国家标准和1项地方标准。培养博士研究生2名，硕士研究生4名。具体研究成果概述如下： 建立了体外培养的永生化绵羊绒毛膜滋养层细胞系（hTERT-STCs），能稳定表达人的端粒酶（hTERT）基因，细胞连续传代一年并持续增殖无衰老的迹象，仍然具有正常的绒毛膜滋养层细胞STCs的生物学特性；成功构建了真核表达质粒pEGFP-C1/enJSRV-env及pEGFP-C1/Hyal2和5个重组干扰质粒，根据荧光定量RT-PCR和Western blot检测结果发现，RNA干扰沉默enJSRV囊膜基因的enJSRV囊膜蛋白表达量与空白和阴性对照组比较显著下降，于是筛选出干扰质粒pcDNA6.2-GW/EmGFPmiR-enJSRV-env-2的干扰效率最强；通过转染pEGFP-C1/enJSRV-env质粒后的绒毛膜滋养层细胞（STCs）及共转染pEGFP-C1/enJSRV-env 与pEGFP-C1/Hyal2后的STCs发生细胞融合的几率增大，平均每个视野分别可以观察到增加了20.44%±7.79%和24.09%±10.65%个绒毛膜滋养层细胞STCs。侵袭性明显增强，阐明enJSRV的ENV蛋白与其受体HYAL2蛋白互作发生细胞融合的机制；利用已构建好的重组表达质粒内源性 pEGFP-C1/enJSRV-env 与外源性pEGFP-C1/exJSRV-env分别体外转染到NIH3T3细胞内，用软琼脂集落形成实验以及裸鼠致瘤实验分别来检测转染不同重组质粒NIH3T3细胞的生长状态，说明了内源性病毒enJSRV 囊膜蛋白（ENV）不能促使NIH3T3细胞发生恶性转化；利用免疫组化法和Western Blot检测到转染重组表达质粒pEGFP-C1/enJSRV-env的绵羊绒毛膜滋养层细胞中，ERK1/2、p38和JNK磷酸化水平均显著高于对照组、转染空质粒pEGFP-C1组及添加抑制因子组（P＜0.05），添加3种抑制剂后，与其相应蛋白激酶的磷酸化明显被抑制，说明内源性enJSRV囊膜蛋白能够激活绵羊绒毛膜滋养层细胞中MAPK信号通路，并且可能通过MAPK信号通路调节绵羊绒毛膜滋养层细胞融合过程。