疟原虫新型分子--TIP对宿主脑型疟疾免疫调节作用及其机制的研究

Malaria causes 216 million clinic cases and 0.7million deaths annually, remaining a major public health problem in world. Cerebral malaria (CM)is a life-threatening complication of malaria. CM mortality rate remains high and the disease causes significant long-term neuro-cognitive deficits in nearly 20% of survivors. The pathogenesis of CM has been studied extensively over several decades. Despite the abundance of information generated on the disease, its pathogenesis remains largely unclear. A unknown protein of malaria parasite --Q813H7 possesses homologous amino acid sequence with that of human T cell immunomodulatory protein (hTIP) and termed as Plasmodium TIP (pTIP). However, its function has remained elusive. In our previous study, the Western blot and RT-PCR experiments revealed the presence of pTIP in schizonts,gametocytes and ookinetes. The expression of pro-inflammatory cytokines decreased and that of Treg increased in the mouse model of CM, as we blockaded pTIP with polyclonal anti-TIP antibody. Those results may suggest that pTIP presents in malaria parasites and may posses a immunomodulatory effect on the genesis and development of CM. We therefore postulates that pTIP may be an essential excretory/secretory protein in the plasmodium lifecycle, especially in the asexual prolification stage and may possess immunomodulatory effect on the host's Th1 response during malaria parasite infection; pTIP may modulate the pro-inflammation response of host to maintain the homeostasis of malaria parasite and host; pTIP may modulate the Th1 immuno-cell subpopulation in the acute infection of malaria parasite by regulating the immuno-response of Th1 immune-cell and the expression of Treg; ?pTIP may mediate the genesis and development of CM by modulating the immuno-response of Th1 immune-cell and Treg. For verification of our hypothesis, we would like to use Plasmodium berghei ANKA and C57BL/6 mice in the experiment to do following works: to get recombined pTIP protein with the prokaryotic expression system and wheat germ cell-free technology and to prepare anti-pTIP monoclonal antibody; to evaluate the characteristic of expression of pTIP in various developmental stage of malaria parasites; to analyze dynamically the effects of pTIP on the genesis and development of CM, including the effects on the blood-brain barrier permeability, RBC and inflammatory cells in microvasculature, the level of CD4+ and CD8+T cell, pro-inflammatory cytokines and anti- inflammatory cytokines, the adhesion molecules and chemotactic factors, Treg, the parasitemia and host survival, as blocking the effect of pTIP with McAb or knocking out the pTIP gene ; to analyze dynamically the effects of pTIP on the target cells-Th1 immuno-cells, as blocking the effect of pTIP with McAb or knocking out the pTIP gene; to analyze the mechanism of the immunomodulation effect of pTIP on Th1 immuno-cells and Treg in vitro after isolating and culturing CD4+T cell and Treg cells.

疟疾每年造成2亿多人感染，70余万人死亡，其中脑疟致死人数占疟疾总死亡人数的80%，20%幸存者留有神经系统的后遗症，脑疟发生发展的机制尚不清楚。疟原虫未知蛋白Q813H7具有与人类TIP同源的氨基酸序列，称为疟原虫TIP（pTIP），其作用尚无报道。我们前期结果证实虫体内有pTIP存在，在多抗阻断pTIP脑疟鼠体内，前炎性因子的表达下降，Treg的表达显著上升。提示pTIP可能对脑疟的发生发展具有免疫调节作用。为探讨该作用及其机制，我们拟用脑疟模型，制备抗pTIP单抗，在单抗阻断和pTIP基因敲除条件下，分析pTIP在虫体不同发育阶段表达及分泌的特点、对脑疟发生发展的作用、对靶细胞-脑疟发生相关的免疫细胞及Treg的作用、体外实验分析pTIP对Th1型免疫细胞及Treg调节作用的机制。以期揭示pTIP对脑型疟疾发生发展的作用机制，为防治脑疟、研发新型高效疫苗提供理论和实验依据。

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