CAJ | 학술논문

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、1000 μmol/L)的Hcy与VSMC孵育,在用Annexin A5(50 μg/ml)或组织因子的单抗(10 μg/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4 h后,100 μmol/L即可诱导VSMC TF蛋白表达升高,阳性率为(14.01±3.72)%,1000 μmol/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100 μmol/L的Hcy作用VSMC,加入Annexin A5或TF的单抗后,在4、8、16 h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VSMC TF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.
목적 연구막련단백A5(Annexin A5)대동형반광안산(Hcy)유도적혈관평활기세포(VSMC)조직인자표체화활화적억제작용.방법 채용조직첩괴법배양인제동맥VSMC,응용α-기동단백(actin)면역조화법감정세포.장불동농도(10、100、500、1000 μmol/L)적Hcy여VSMC부육,재용Annexin A5(50 μg/ml)혹조직인자적단항(10 μg/ml)간예적조건하,류식세포기술(FCM)검측VSMC적조직인자세포막표체,FX생성반응검측VSMC세포배양액조직인자활성,Western blot검측VSMC적조직인자표체.결과 용PBS작위대조적VSMC세포막표면유저수평적TF표체,양성솔위(4.01±2.11)%.Hcy작용4 h후,100 μmol/L즉가유도VSMC TF단백표체승고,양성솔위(14.01±3.72)%,1000 μmol/L시체고봉,양성솔위(37.67±4.96)%.여PBS대조조세포상비,Hcy능현저유도세포TF적표체.용100 μmol/L적Hcy작용VSMC,가입Annexin A5혹TF적단항후,재4、8、16 h균능유효억제TF적세포막표체화배양액중TF활성.불동농도적Annexin A5능현저억제Hcy유도적VSMC TF단백적표체.결론 Annexin A5능억제Hcy유도적VSMC조직인자적표체화활화,대차작용과정적심입연구,가능위방치관상동맥죽양반괴혈전적형성화발전제공신적계궤.
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 pbosphatidylserine. 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.