摘要
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(25μmol/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(50μmol/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。
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 p｜asmid encoding glutathione S-transferase （GST）-p21-binding domain of p21-activated kinase 1 （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 p^mol/L）. Wound- healing assay and Transwell assay were performed to observe the changes of C. pn infection-induced VSMCs migration with or without pre-incubation of Racl inhibitor NSC23766 （50 Izmol/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. Rael 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 sig- nificantly reduced and was lower than that in C. pn infection group. CONCLUSION ： C. pn infection induces the migration of VSMC possibly through stimulating Racl activity via PI3K activation.
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2013年第2期236-241,共6页
Chinese Journal of Pathophysiology
基金
国家自然科学基金资助项目(No.30971225)
教育部科学技术研究重点项目(No.206008)
高等学校博士点基金资助项目(No.20111202110011)
