摘要
目的探讨融合蛋白TAP-SSL5对血小板微粒(platelet microparticles,PMPs)与人单核细胞株THP-1细胞结合及Mac-1活化的影响。方法以二磷酸腺苷(adenosine diphosphate,ADP)激活人血小板并获取PMPs。采用流式细胞仪(flow cytometry,FCM)及PE标记的抗CD62P单克隆抗体、FITC标记的Annexin V检测PMPs,以FITC标记的抗CD41单克隆抗体和PE标记的抗CD154(CD40L)单克隆抗体检测PMPs的表面特征。采用JC-1试剂盒检测血小板线粒体膜电位。采用FCM检测PMPs与THP-1细胞的结合,以及PMPs诱导THP-1细胞表面Mac-1(CD11b/CD18,αMβ2)的活化情况,并研究TAP-SSL5的干预作用。结果 PMPs呈现CD62P和Annexin V双阳性,且CD41和CD40L的阳性率分别达到50.8%和44.0%。JC-1检测显示,ADP对血小板线粒体膜电位无明显影响(P>0.05)。PMPs与THP-1细胞的结合率为(24.80±5.16)%,PMPs诱导THP-1细胞Mac-1的活化率为(21.17±5.92)%,THP-1细胞经10 mg/L TAP-SSL5预处理后,PMPs的结合率下降至(13.67±2.15)%(P<0.05),Mac-1的活化率下降至(0.99±0.62)%(P<0.01)。结论 TAPSSL5可抑制PMPs与THP-1细胞的结合及THP-1细胞表面Mac-1的活化。
Objective To investigate the effect of anticoagulant and anti-inflammatory fusion protein TAP-SSL5 （tick anticoagulant peptide and staphylococcal superantigen like protein-5） on the binding of platelet microparticles （PMPs） to human acute monocytic leukemia cell line THP-1 and the activation of Mac-1. Methods PMPs were generated by adenosine diphosphate （ADP） activating human platelets. Flow cytometry （FCM） was used to identify PMPs by PE labeled mouse anti-human CD62P monoclonal antibody and FITC-Annexin V, and to check the activation and phosphatidylserine （PS） by FITC labeled mouse anti-human CD41 monoclonal antibody and PE labeled mouse anti-human CD151 monoclonal antibody （CD40L）. The mitochondrial membrane potential in human platelets was checked with JC-1 kit. The binding rates of PMPs to THP-1 cells and the conformation change of Mac-1 （CD11b/CD18, αMβ2） after co-incubation with PMPs were assayed by FCM. Results Both CD62P and PS were positive on PMPs. The positive rates of CD41 and CD40L on the ADP-induced PMPs were 50.8% and 44.0% respectively. While there was no significant change on the mitochondrial membrane potential in ADP activated platelets （P〉0.05）. The binding rates of PMPs to THP-1 cells and the activation rates of Mac-1 on THP-1 cells were （24.80±5.16）% and （21.17±5.92）% respectively, which decreased to （13.67±2.15）% and （0.99±0.62）% after the THP-1 cells were pre-incubated with 10 mg/L TAP-SSL5 （P〈0.05 and P〈0.01）. Conclusion TAP-SSL5 directly inhibits the binding of PMPs to THP-1 cells, and subsequently inhibits the activation of Mac-1 on THP-1 cells.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2014年第9期864-867,共4页
Journal of Third Military Medical University
基金
国家自然科学基金(81270362)
国家重大新药创制课题(2013ZX09103003-001)~~
