表皮生长因子受体通路调控隐球菌穿越血脑屏障的分子机制研究

The critical step of cryptococcal central nervous system infection is the breakthrough of host blood-brain-barrier (BBB). The molecular mechanism has become a hot point in currently research. Our previous study displayed that annexin A2 of brain microvascular endothelial cells played an important role in blood-brain-barrier invasion of Cryptococcus neoformans. Down-regulation of Annexin A2 enhanced the efficiency of cryptococcal adhesion and penetration to host brain microvascular endothelial cells, which might be closely related to the.epidermal growth factor receptor (EGFR) expression or reutilization. On the basis, we further found that application of a specific inhibitor of EGFR was significantly inhibited the efficiency of the pathogen to across the BBB. We speculated that EGFR signaling pathway might be essential for the interaction of the pathogen and BBB. The molecular mechanism needs to be further clarified. In this study, we try to detect how EGFR and its downstream signaling pathway participate in the process of cryptococcal crossing the BBB via in vitro BBB model.and animal infection models, combined with functional genomic techniques such as RNA interference, tissue-specific gene knockout, immunofluorescence, and mass spectrometry. Our study will provide new theoretical basis for exploring specific intervention targets against cryptococcosis.

隐球菌侵染宿主中枢神经系统的关键环节在于宿主血脑屏障的突破，其分子机制尚不甚明确，已成为当前学界的研究热点。通过前期研究，我们发现：脑微血管内皮细胞的膜联蛋白A2可能是隐球菌穿越血脑屏障的重要调控分子，该蛋白下调表达可增进隐球菌对脑微血管内皮细胞的粘附及穿透效率，其潜在的分子机制可能与内皮细胞膜表面表皮生长因子受体（EGFR）的表达与回收密切相关。在此基础上，我们进一步发现，应用EGFR特异性抑制剂能显著抑制隐球菌穿越血脑屏障的效率，因此推测EGFR及其信号通路可能是参与隐球菌穿越血脑屏障的关键调控通路，其内在的分子机制亟待进一步明确。 本研究拟应用体外血脑屏障模型和动物感染模型，结合RNA干扰、组织特异性基因敲除、免疫荧光、蛋白质谱等方法深入研究EGFR及下游信号转导通路在隐球菌穿越血脑屏障过程中潜在的功能效应和分子机制，为探索隐球菌脑膜脑炎的新型特异性药物干预靶点提供理论依据。

病原体穿越血脑屏障是隐球菌感染中枢神经系统的关键，隐球菌与脑微血管内皮细胞的相互作用关系是阐明隐球菌突破宿主血脑屏障防御分子机制的关键环节。本课题组在前期研究发现 AnxA2 蛋白在隐球菌侵袭宿主血管内皮细胞的过程中发挥保护作用，该种作用很可能依赖于宿主细胞表皮生长因子受体通路来完成调节。本课题对于EGFR信号通路调控隐球菌穿越血脑屏障分子机制进行了系统的研究，研究发现隐球菌金属蛋白酶Mpr1可以调节宿主细胞HB-EGF的表达水平，HB-EGF的表达会损坏隐球菌侵染宿主血脑屏障的胞外连接水平并且加强隐球菌穿越血脑屏障的能力。而HB-EGF/EGFR信号通路对于隐球菌穿越血脑屏障的调控主要依赖信号蛋白磷酸化级联反应而完成调节作用。这些研究将有利于建立新型的隐球菌脑膜炎干预策略。