N-WASP 磷酸化在肺炎衣原体感染促进血管新生中的作用
王海伟，王蓓蓓，张利军，马路，刘静雅，张丽莙
基金项目：国家自然科学基金资助项目(No.30971225、No.81300206)；高等学校博士点基金项目（No.
20111202110011）
作者简介：王海伟（1986 年-），女，研究生，主要研究方向：动脉粥样硬化
通信联系人：张丽莙（1963 年-），女，教授，主要研究方向：动脉粥样硬化. 

 （天津医科大学基础医学院生理学与病理生理学系，天津300070）
摘要：目的 探讨N-WASP 磷酸化在肺炎衣原体（C.pn）感染诱导血管新生中的作用及其可能机制。方法 C.pn 增殖培养后感染人血管内皮细胞（VEC），免疫荧光染色确认感染成功；Western blot 检测C.pn 感染的VEC 内N-WASP 的磷酸化水平；CCK-8 检测N-WASP 特异性抑制剂Wiskostain 对VEC 活力的影响；免疫荧光实验检测工作浓度的Wiskostain 在使用时间内对C.pn 感染率的影响；C.pn 感染Wiskostain（5 μM）预处理的VEC 后，Western blot检测N-WASP 的磷酸化水平，Capillary tube formation 实验观察各组VEC 形成新生血管能力的变化。结果 在荧光显微镜下，感染的VEC 胞浆内可见典型的C.pn 包涵体。C.pn 感染VEC10 h 和24 h 后N-WASP 的磷酸化水平均明显上调且高于正常对照组；经Wiskostain 预处理后，C.pn 感染上调N-WASP 磷酸化水平的作用则明显被抑制（P < 0.05）。Capillary tube formation 实验结果显示，C.pn 感染VEC 16 h 后，其所形成的微管腔节点数明显多于对照组（P < 0.05）；经Wiskostain 预处理后，C.pn 感染促进微管腔节点形成的作用则被显著削弱，几乎不能形成微管腔结构（P < 0.05）。结论 C.pn 感染可能通过诱导N-WASP 磷酸化促进VEC 形成新生血管。
关键词：肺炎衣原体；血管新生；N-WASP；动脉粥样硬化
中图分类号：R363
Roles of N-WASP phosphorylation in angiogenesis promoted
by Chlamydia pneumoniae infection
WANG Haiwei, WANG Beibei, ZHANG Lijun, MA Lu, LIU Jingya, ZHANG Lijun
 (Department of Physiology and Pathophysiology, School of Basic Medical Sciences, Tianjin Medical University, Tianjin 300070, China)
Abstract: Objective To explore the roles of N-WASP phosphorylation in Chlamydia pneumoniae(C.pn) infection-induced angiogenesis and the possible mechanisms in this process. Method VECs were infected with C.pn after its propagation. The successful infection of C.pn with VECs was identified by immumofluorescence. Western blot was performed to determine N-WASPphosphorylation level. The cytotoxicity of Wiskostain was assessed by a CCK-8 assay. The effects of Wiskostain (5 μM) on C.pn infection of VECs were determined by immumofluorescence. After VECs were pretreated with Wiskostain (5 μM), Western blot was used to detect the changes in N-WASP phosphorylation levels in VECs from each group, and capillary tube formation assay was performed to observe the changes in the abilities of infected VECs to form new blood vessels.
Results The typical C.pn inclusions were observed in the cytoplasm of the infected VECs under a fluorescence microscope. Western blot results showed that the phosphorylation levels of N-WASP were signigicantly increased 10 h and 24 h after infection, and were higher than that in control VECs, which was significantly inhibited when VECs were pretreated with Wiskostain (P < 0.05).The results from capillary tube formation assay showed that the number of network branch points