金黄色葡萄球菌超抗原样蛋白-5抑制人脐血源性内皮祖细胞黏附功能及其机制研究  

Staphylococcal superantigen-like protein-5 inhibits adhesion of human umbilical cord blood-derived endothelial progenitor cells to P-selectin-coated surface

作　　者：梁华[1] 曲小龙[1] 胡厚源[1] 宋治远[1] 程彦[1] 张静[1] 

机构地区：[1]第三军医大学西南医院心血管内科,重庆市介入心脏病学研究所,重庆400038

出　　处：《第三军医大学学报》2011年第6期545-549,共5页Journal of Third Military Medical University

基　　金：国家高技术研究发展计划基金(863计划,2009AA02Z115);国家自然科学基金(30871059);重庆市自然科学基金(CSTC2009BB5018);第三军医大学回国人员启动基金(2008XG05)~~

摘　　要：目的研究金黄色葡萄球菌超抗原样蛋白-5(staphylococcal superantigen-like prote in-5,SSL5)与人脐血源性内皮祖细胞(endothelial progen itor cells,EPCs)表面P-选择素糖蛋白配体-1(P-selectin glycoprote in ligand-1,PSGL-1)的结合情况,及其对内皮祖细胞黏附功能的影响。方法从金黄色葡萄球菌NCTC 8325菌株的基因组中,扩增ssl5基因,并进行重组SSL5蛋白表达载体的构建。采用密度梯度离心法分离得到脐血中的单个核细胞并进行体外培养,对贴壁细胞在激光共聚焦显微镜下观察其摄取乙酰化低密度脂蛋白(D iI-acLDL)和结合荆豆凝集素(FITC-UEA-1)的情况。以流式细胞仪分析SSL5与EPCs表面PSGL-1的结合情况;以calce in-AM负载EPCs后,定量分析SSL5对EPCs在P-选择素包被表面黏附的抑制作用。结果 D iI-acLDL/FITC-UEA-1双染阳性的细胞为EPCs。PSGL-1在EPCs表面有较丰富的表达,阳性细胞率为76.6%。SSL5与EPCs的结合随着SSL5浓度的增加而显著升高;并且,SSL5可竞争性抑制抗PSGL-1单克隆抗体(KPL-1)与EPCs的结合。SSL5可显著抑制EPCs在P-选择素表面的黏附,终浓度为30 mg/L的SSL5对EPCs在P-选择素表面黏附的抑制率已接近10 mg/L的KPL-1的效应,两者与空白对照组比较,差异有统计学意义(P<0.01)。结论 SSL5可与EPCs表面的PSGL-1结合,而抑制EPCs在P-选择素表面的黏附,提示SSL5可能通过抑制EPCs与损伤内皮或激活的血小板之间的黏附,进而抑制EPCs对损伤内皮的修复作用。Objective To investigate the binding of staphylococcal superantigen-like protein-5（SSL5） to P-selectin glycoprotein ligand-1（PSGL-1） on human umbilical cord blood-derived endothelial progenitor cells（EPCs） and the inhibitive effect of SSL5 on the adhesion of EPCs to P-selectin-coated surface.Methods SSL5 gene was amplified from the genome of Staphylococcus aureus NCTC 8325 and cloned into a vector for expressing recombinant SSL5 protein.Mononuclear cells were isolated from human umbilical cord blood by density gradient centrifugation and then cultured in vitro.The acetylated low-density lipoprotein（DiI-acLDL） uptake and Ulex europaeus agglutinin（FITC-UEA-1） interaction of the adherent cells were observed using a laser scanning confocal microscope.The expression of PSGL-1 on EPCs and the binding between SSL5 and PSGL-1 on EPCs were examined using a fluorescence-activated cell sorter.Calcein-AM-labeled EPCs were cultured with different concentrations of SSL5（0,1,3,10 and 30 mg/L）,and the inhibitory effect of SSL5 on the adhesion of EPCs to P-selectin-coated surface was determined quantitatively.Results Almost all EPCs were positive in DiI-acLDL and FITC-UEA-1 staining.PSGL-1 was highly expressed on EPCs with a positive cell rate of 76.6%.The binding of SSL5 to PSGL-1 on EPCs was significantly and positively correlated with SSL5 concentration.SSL5 competitively inhibited the binding of anti-PSGL-1 monoclonal antibody（KPL-1） to EPCs.SSL5 significantly suppressed the adhesion of EPCs to P-selectin-coated surface,and 30 mg/L SSL5 presented similar inhibition effect to 10 mg/L KPL-1,showing significant difference as compared with the control group（P0.01）.Conclusion SSL5 can bind to PSGL-1 on EPCs and inhibit the adhesion of EPCs to P-selectin-coated surface,which indicates that SSL5 may hinder the repair effect of EPCs on blood vessels by inhibiting the binding of EPCs to injured vascular endothelia or activated blood platelets.

关 键 词：金黄色葡萄球菌超抗原样蛋白-5 P-选择素糖蛋白配体-1 内皮祖细胞 黏附 

分 类 号：R378.11[医药卫生—病原生物学] R394.3[医药卫生—基础医学]