肺炎衣原体感染经由IQGAP1通过削弱VE-钙粘素相关的血管内皮细胞间粘附连接促进单核细胞跨内皮迁移

Transendothelial migration (TEM) of monocytes from blood to the intima is a initial step during the pathogenesis of atherosclerosis. Up to now, few researchers have examined the mechanism of Chlamydia pneumoniae ( C.pn) infection-induced TEM of monocytes by investigating the effect of C.pn infection on vascular endothelial adherens junctions (AJ). In our research, after we examine the effect of C.pn infection on the interendothelial AJ by a immunofluorescence technique, we will explore the regulatory role of IQ domain GTPase-activating protein 1 (IQGAP1) in this process: In endothelial cells, after C.pn infection, Western blot will be performed to determine the protein expression levels of IQGAP1 and vascular endothelial cadherin (VE- cadherin), and the immunofluorescence technique will be used to determine the colocalization of IQGAP1 and VE- cadherin at the AJ. Co-immunoprecipitation assay will be performed to demonstrated that IQGAP1 forms a complex with VE- cadherin in the infected endothelial cells. Then, we will change the expression levels of IQGAP1 by use of the technologies of gene transfection and RNA interference to investigate the effect of C.pn infection on the AJ by the immunofluorescence technique. Moreover, we will further examine the effect of infection of endothelial cells with C.pn on TEM of monocytes using the TEM assay in vitro after we change the expression levels of IQGAP1 by use of the technologies of gene transfection and RNA interference. By clarifying the effect of C.pn infection on the interendothelial AJ and the related mechanism, our research will not only present a new mechanism by which C.pn infection promotes TEM of monocytes, but also find a new strategy for preventing atherosclerosis.

单核细胞跨内皮迁移入动脉内膜是动脉粥样硬化（AS）发病的起始环节。目前鲜有文献从肺炎衣原体（C.pn）感染影响血管内皮细胞（EC）间连接的角度阐述其促进单核细胞跨内皮迁移的机制。我们在利用免疫荧光技术观察C.pn感染对EC间粘附连接（AJ）影响的基础上，探讨IQGAP1在C.pn感染对AJ影响中的调控作用：C.pn感染EC后，Western blot和免疫荧光分别观察IQGAP1和VE-钙粘素的蛋白表达水平及其在AJ的共定位，免疫共沉淀鉴定IQGAP1/VE-钙粘素复合物的存在；应用基因转染和RNA干扰技术分别改变IQGAP1表达后，免疫荧光观察C.pn感染对AJ的影响；此外，改变IQGAP1表达后通过体外跨内皮迁移实验观察单核细胞跨内皮迁移能力的变化。本项目通过阐明C.pn感染对EC间AJ的影响及作用机制，不仅揭示C.pn感染促进单核细胞跨内皮迁移的新机制，同时也为预防AS提供新策略。

单核细胞跨内皮迁移入动脉内膜是动脉粥样硬化发病的起始环节。目前鲜有文献从肺炎衣原体（C.pn）感染影响血管内皮细胞（VEC）通透性的角度阐述其促进单核细胞跨内皮迁移的机制。本课题利用C.pn体外感染人脐静脉内皮细胞系Ea.hy926细胞模型，跨内皮迁移实验观察C.pn感染VEC对单核细胞跨内皮迁移的影响，结果显示，C.pn感染VEC可促进单核细胞跨内皮迁移。为阐明C.pn感染VEC促进单核细胞跨内皮迁移的机制，我们利用跨内皮电阻（Transendothelial electrical resistance, TEER）实验检测C.pn感染对VEC通透性的影响，发现C.pn感染VEC后VEC通透性明显增加，而这与C.pn感染促进单核细胞跨内皮迁移有关。C.pn感染如何增加VEC通透性？VE-cadherin内吞在VEC通透性增加中发挥重要作用。因此，我们利用免疫荧光实验观察C.pn感染对VE-cadherin内吞的影响，结果表明，C.pn感染可明显促进VE-cadherin内吞。异硫氰酸荧光素标记的牛血清白蛋白（Fluorescein-isothiocyanate labeled bovine serum albumin，FITC-BSA）实验结果表明，C.pn感染可能通过促进VE-cadherin内吞而增加VEC通透性。VE-cadherin的磷酸化水平与VE-cadherin内吞密切相关。Western blot结果显示，C.pn感染可促进VE-cadherin（Y658）酪氨酸磷酸化，这可能是通过激活VEC内Src激酶来实现的。应用Src激酶选择性抑制剂PP2预处理VEC后，C.pn感染增强VE-cadherin酪氨酸磷酸化的作用被明显抑制，同时，C.pn感染促进VE内吞的效应也被显著削弱；TEER实验结果显示，PP2可明显抑制C.pn感染增加VEC通透性的作用；跨内皮迁移实验结果表明，VE-cadherin酪氨酸磷酸化在C.pn感染促进单核细胞跨内皮迁移中发挥重要作用。综上，本项目证实了C.pn感染可能通过激活VEC内Src激酶促使VE-cadherin发生酪氨酸磷酸化，并促进VE-cadherin内吞，进而增加VEC通透性而诱导单核细胞跨内皮迁移，揭示了C.pn感染VEC促进单核细胞跨内皮迁移的新机制，同时也为预防动脉粥样硬化提供新策略。