摘要
目的研究膜联蛋白A5（Annexin A5）对同型半胱氨酸（Hcy）诱导的血管平滑肌细胞（VSMC）组织因子表达和活化的抑制作用。方法采用组织贴块法培养人脐动脉VSMC，应用α-肌动蛋白（actin）免疫组化法鉴定细胞。将不同浓度（10、100、500、1000μmol／L）的Hcy与VSMC孵育，在用Annexin A5（50μg／ml）或组织因子的单抗（10μg/ml）干预的条件下，流式细胞技术（FCM）检测VSMC的组织因子细胞膜表达，FX生成反应检测VSMC细胞培养液组织因子活性，Western blot检测VSMC的组织因子表达。结果用PBS作为对照的VSMC细胞膜表面有低水平的TF表达，阳性率为（4．01±2．11）％。Hcy作用4h后，100μmol／L即可诱导VSMCTF蛋白表达升高，阳性率为（14．01±3．72）％，1000μmol／L时达高峰，阳性率为（37．67±4．96）％。与PBS对照组细胞相比，Hcy能显著诱导细胞TF的表达。用100μmol／L的Hcy作用VSMC，加入Annexin A5或TF的单抗后，在4、8、16h均能有效抑制TF的细胞膜表达和培养液中TF活性。不同浓度的Annexin A5能显著抑制Hcy诱导的VSMCTF蛋白的表达。结论Annexin A5能抑制Hcy诱导的VSMC组织因子的表达和活化，对此作用过程的深入研究，可能为防治冠状动脉粥样斑块血栓的形成和发展提供新的契机。
Objective The expression of tissue factor （TF） in vascular smooth muscle cells （VSMCs） plays an important role in the pathogenesis of artherosclerosis （AS） and thrombosis. Hyperhomocysteinemia is a risk factor for AS. Annexin AS, a calcium-dependent anionic-phospholipid-binding protein has anticoagulant effect mediated by its interaction with phosphatidylserine. We investigated the effects of Annexin A5 on homocysteine （Hcy） -induced TF expression and activity in VSMCs. Methods Human umbilical artery VSMCs were cultured by tissue explanting method, incubated with Hcy at various concentrations in the absence and presence of Annexin A5 （ 50 μg/ml）/ mono-TFAb （ 10 μg/ml ）. Flow Cytometry （FCM） was used to detect the expression of TF on the surface of VSMCs. Protein expression of TF was detected by Western blot. Determination of TF activity by factor Xa generation. Results The expression level of TF protein on the surface of the resting VSMCs was low [ the positive rate was （4.01 ± 2.11 ）% ] and could be upregulated by Hcy [ peaked at 1000 μmol/L, the positive rate was （37.67 ± 4.96） % ]. Annexin AS, as well as mono-TFAb could significantly inhibit Hey-induced TF membrane expression, release activity and protein expression. Conclusions These results suggest that Annexin A5 could inhibit Hcy-induced expression and activity of TF in VSMCs as well as TF release of VSMCs. Annexin A5 might play an active role on attenuating AS and reducing coronary thrombosis by inhibiting TF pathway.
出处
《中华心血管病杂志》
CAS
CSCD
北大核心
2009年第11期1039-1043,共5页
Chinese Journal of Cardiology
基金
基金项目：国家自然科学基金资助项目（30200109）
