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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 effect of staphylococcal superantigen-like prote-5 (SSL5) on the surface of P-selectin glycoprotein ligands on human umbilical cord blood-derived endothelial progenitor cells (EPCs) 1 (P-selectin glycoprotein ligand-1, PSGL-1), and its effect on the adhesion function of endothelial progenitor cells. Methods The ssl5 gene was amplified from the genome of Staphylococcus aureus NCTC 8325 and the recombinant SSL5 protein expression vector was constructed. Mononuclear cells from umbilical cord blood were isolated by density gradient centrifugation and cultured in vitro. The adherent cells were observed under laser confocal microscopy for uptake of acetylated low density lipoprotein (D iI-acLDL) and binding of Vitexin (FITC-UEA-1). The binding of SSL5 to PSGL-1 on the surface of EPCs was analyzed by flow cytometry. After EPCs were loaded with calcein-AM, the inhibitory effects of SSL5 on the adhesion of EPCs to P-selectin-coated surfaces were quantitatively analyzed. Results D iI-acLDL / FITC-UEA-1 double-stained cells were EPCs. PSGL-1 was abundantly expressed on the surface of EPCs, with a positive rate of 76.6%. The binding of SSL5 to EPCs was significantly increased with the increase of the concentration of SSL5, and SSL5 competitively inhibited the binding of anti-PSGL-1 monoclonal antibody (KPL-1) to EPCs. SSL5 significantly inhibited the adhesion of EPCs on P-selectin surfaces, and the effects of SSL5 at a final concentration of 30 mg / L on KPL-1 with EPCs adherence on P-selectin surface approaching 10 mg / L, both Compared with the blank control group, the difference was statistically significant (P <0.01). Conclusion SSL5 binds to PSGL-1 on the surface of EPCs and inhibits the adhesion of EPCs on the surface of P-selectin, suggesting that SSL5 may inhibit the adhesion of EPCs to injured endothelium or activated platelets and thereby inhibit the repair of injured endothelium by EPCs effect.