东方田鼠补体杀伤日本血吸虫童虫特性与机制研究

Schistosomiasis is a serious zoonotic parasitic diseases in the world，urgently need to develop new techniques for prevention and control. Microtus fortis is a exclusive and specific animal model of anti-Schistosomasis japonicum in China, the research on its natural resistance mechanism has theoretical and practical significance. In the previous study, two closed colonies of Microtus fortis had been established and the killing effect of serum complement against newly transformed schistosomula were confirmed in vitro. This project is intended to further analyse the role of three activation pathways in complemnet cascade and the killing effect of serum complement against liver-stage schistosomula in culturing system. Based on the effect of complement against schistosomula in the early stage of infection confirmed by inhibitors in vivo, here we proposed to construct a recombinant RNAi Lentivirus specific to the complement C3 molecule for interfereing the expression and activity of complement molecules in the vole, to observe its sensitivity to infection, and finally to elucidate the role and mechanism of complement in Microtus fortis resistance to schistosoma japonicum worm. The complement hemolysis test were used to analyze the inhibiting effect of the complement inhibitors of Schistosoma japonicum on complement molecules of Microtus fortis, and to interpret the relation between the resistance of Microtus fortis and the inhibition of complement activity by Schistosoma japonicum. The results of the study will provide a better understanding of the role and mechanism of complement of Microtus fortis resistance to schistosoma japonicum, which will help to understand the anti-schistosomiasis characteristics of the vole and also help to discover new techniques and strategies for schistosomiasis prevention and control.

血吸虫病人畜共患，全球流行,危害严重，急需开拓防治新技术。东方田鼠是我国独有且特异的抗血吸虫病模型动物,其抗性机制研究具有理论价值和现实意义。本项目在前期建立了东方田鼠实验动物封闭群的基础上，证实了东方田鼠血清补体对血吸虫童虫具有显著杀伤作用。本申请拟进一步分析东方田鼠血清补体对血吸虫肝期童虫的杀伤作用，研究3条补体激活通路在东方田鼠补体对血吸虫杀伤中的作用。通过制备东方田鼠补体C3分子特异的重组RNA干扰慢病毒抑制东方田鼠补体活性，验证补体在东方田鼠抗血吸虫特性中的作用。利用补体溶血试验研究分析日本血吸虫补体抑制蛋白对东方田鼠补体分子的抑制作用，阐述东方田鼠抗性和血吸虫在其体内未能逃避补体免疫应答的相关性。这些研究结果将更好地解析东方田鼠补体在其抗性中的作用和机制，有助于理解东方田鼠抗血吸虫特性，也有助于发现新的防治血吸虫病的技术和策略。

血吸虫病人畜共患，全球流行，危害严重。东方田鼠是我国独有且特异的抗血吸虫病模型动物,其抗性机制研究具有理论价值和现实意义。本项目在前期建立了东方田鼠实验动物封闭群的基础上，进一步明确了日本血吸虫在东方田鼠体内的消亡主要在感染后早期（1-3d和8-12d）。东方田鼠补体活性在感染后呈现曲线变化，与感染和童虫消亡的过程相关，肺脏和肝脏组织的补体活性与童虫移行有关。感染后，肝组织C3、C5、C9等补体分子表达升高，血清中C3和C4含量呈上升趋势。东方田鼠补体分子在肝脏中的表达水平最高，与小鼠等其它物种相似。东方田鼠血清孵育的尾蚴转化童虫死亡率为85.50%，灭活补体后童虫死亡率为60.99%。扫描电镜观察显示对照组童虫体表小棘密集，虫体轮廓清晰；血清孵育组童虫轮廓不完整，体表突出的嵴和凹窝有脱落现象。免疫荧光表明东方田鼠血清孵育的童虫体表有MAC沉积。用C1qBP处理抑制补体经典途径后童虫死亡率为16.99%，对照组C1qBP蛋白孵育的童虫死亡率为8.14%。抑制旁路途径和凝集素途径后童虫死亡率差异不显著。东方田鼠正常血清和补体灭活血清对10d肝期童虫的杀伤效果差异不显著。日本血吸虫补体抑制蛋白rSjTOR-ed1和rSjC1qbp均呈浓度依赖性抑制补体溶血反应，溶血抑制率分别为59.47%和77.88%。克隆并鉴定了东方田鼠补体分子C3和C5基因，序列比对结果与草原田鼠的同源性大于90%。设计并合成了C3和C5基因特异的各三种AAV8型腺相关病毒。rAAV8-shRNA4-MfC3可诱导小鼠肝脏C3表达量降低20.03%-62.70%。东方田鼠尾静脉注射rAAV-MFC3-ShRNA4/MF病毒后感染尾蚴，10d虫体数为75.67±46.72，虫体发育率为10.21%，分别是对照组的4.47倍和2.45倍，肝脏C3转录水平降低26.61%。注射rAAV-MFC5-ShRNA4/MF重组病毒后，14d虫体发育率为2.20%，是对照组的1.61倍，肝组织C5转录降低37.80%。腹腔注射CVF后8d童虫回收率极显著提高（23.20% vs 8.54%）。结果表明东方田鼠补体分子在感染后早期阶段中的杀虫作用中具有重要作用。研究结果可解释东方田鼠补体在其抗性中的作用和机制。共发表论文13篇，其中SCI论文6篇，获得发明专利4项，培养硕士研究生6名。