AIM: To determine the role of Racl activation in the migration of vascular smooth muscle cells ( VSMCs) induced by Chlamydia pneumoniae (C. pn) infection and to investigate the effect of phosphatidylinositol 3-kinase (PI3K) on Racl activation during the process. METHODS; The recombinant plasmid encoding glutathione 5-transferase (GST) -p21 -binding domain of p21-activated kinase l(PBD) fusion protein was transformed into the competent bacteria to induce the expression of the fusion protein. The fusion protein was purified. GST-pull down assay was performed to evaluate the activity of Racl in C. pn-infected VSMCs pretreated with or without PI3K inhibitor LY294002 (25 μmol/L) . Wound-healing assay and Transwell assay were performed to observe the changes of C. pn infect ion-induced VSMCs migration with or without pre-incubation of Racl inhibitor NSC23766 (50 μmol/L) . RESULTS: Enough and biologically active GST-PBD fusion protein was obtained after purification. The activity of Racl in VSMCs infected with C. pn for 24 h increased significantly and was higher than that in control group. Racl activation induced by C. pn infection was inhibited by the pre-treatment of VSMCs with LY294002. The migration ability of C. pn-infected VSMCs pre-incubated with NSC23766 was significantly reduced and was lower than that in C. pn infection group. CONCLUSION: C. pn infection induces the migration of VSMCs possibly through stimulating Racl activity via PI3K activation.%目的:研究Rac1活化在肺炎衣原体(C.pn)感染诱导血管平滑肌细胞(VSMCs)迁移中的作用及磷脂酰肌醇3-激酶(PI3K)对其活化的影响.方法:谷胱甘肽巯基转移酶(GST)-p21活化激酶1 p21结合结构域(PBD)即GST-PBD重组质粒转化感受态细菌,诱导融合蛋白表达并纯化;GST-pull down实验检测C.pn感染VSMCs后Rac1活性的变化;PI3K特异性抑制剂LY294002 (25 μmol/L)预处理VSMCs,GST-pull down实验检测Rac1活性的变化;Rac1特异性抑制剂NSC23766(50 μmol/L)预处理VSMCs,wound-healing 实验和Transwell 实验观察VSMCs迁移能力的变化.结果:重组质粒转化感受态细菌后,经诱导表达和纯化,得到足量有效的GST-PBD融合蛋白;GST-pull down实验结果显示,C.pn感染VSMCs后Rac1活性增强且显著高于正常对照组(P<0.05);LY294002预处理VSMC后,C.pn感染诱导的Rac1活性明显下降(P<0.05);细胞迁移实验结果显示,NSC23766预处理的 C.pn感染组细胞迁移能力明显低于单纯感染组(P<0.05).结论:C.pn感染可能通过PI3K激活Rac1,从而诱导VSMCs迁移.
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