摘要
目的:研究抗炎、抗凝双效融合蛋白蜱抗凝血肽(TAP)-金黄色葡萄球菌超抗原样蛋白5(SSL5)对激活的血小板与人淋巴细胞结合作用的影响。方法:采用免疫磁珠分选法筛选人外周血总淋巴细胞;CCK-8法检测TAP-SSL5对细胞活力的影响;流式细胞术检测Jurkat细胞(人外周血白血病T细胞株)表面CD162(PSGL-1)的表达及TAP-SSL5对小鼠抗人CD162单抗(KPL-1)与Jurkat细胞结合的抑制作用。以20μmol/L ADP激活人血小板,流式细胞术检测血小板与Jurkat细胞或人淋巴细胞的结合情况,并研究TAP-SSL5的干预作用。结果:30mg/L及以下浓度的TAP-SSL5对Jurkat细胞的活力无明显影响。流式细胞术检测显示,10 mg/L的TAP-SSL5能显著抑制KPL-1与Jurkat细胞的结合;20μmol/L ADP激活的血小板与Jurkat细胞或淋巴细胞的结合率分别为(11.86±4.49)%和(8.32±1.00)%;细胞经10 mg/L TAP-SSL5预先处理后,结合率分别降至(6.73±2.71)%和(5.51±0.70)%,差异有统计学意义。结论:TAP-SSL5可与淋巴细胞表面的PSGL-1结合,从而抑制激活的血小板与人淋巴细胞的结合,这可能是抗炎、抗凝双效融合蛋白TAP-SSL5发挥其抗炎作用的机制之一。
AIM： To study the effect of tick anticoagulant peptide-staphylococcal superantigen like protein 5 （TAP-SSL5）, an anti-inflammatory and anticoagulant fusion protein , on the binding of activated platelets to human lym-phocytes.METHODS：Human periphery lymphocytes were isolated by magnetic activated cell sorting （MACS）.The toxic-ity of TAP-SSL5 on the viability of Jurkat cell was assessed by CCK-8 assay.Flow cytometry was applied to detect the ex-pression of CD162 （PSGL-1） on the Jurkat cells （human peripheral blood leukemia T lymphocyte cell line ） and the inhibi-tory effect of TAP-SSL5 on the binding of mouse anti-human CD162 monoclonal antibody （KPL-1） to Jurkat cells.Platelets were activated by ADP at concentration of 20μmol/L, the binding rates of activated platelets to Jurkat cells or human lym-phocytes were assayed by flow cytometry .RESULTS：The concentration of TAP-SSL5 below 30 mg/L didn’ t affect the vi-ability of Jurkat cells .TAP-SSL5 at 10 mg/L competitively inhibited KPL-1 binding to Jurkat cells .The binding rates of activated platelets to Jurkat cells or lymphocytes were （11.86 ±4.49）% and （8.32 ±1.00）%, respectively, which de-creased to （6.73 ±2.71）%and （5.51 ±0.70）%after the Jurkat cells and lymphocytes were pre-incubated with 10 mg/L TAP-SSL5 （P 〈0.05）.CONCLUSION：TAP-SSL5 binds to PSGL-1 expressed on lymphocyte surface and directly in-hibits the binding of activated platelets to human lymphocytes , which may be one of the anti-inflammatory mechanisms of TAP-SSL5.
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2015年第1期23-27,共5页
Chinese Journal of Pathophysiology
基金
国家重大新药创制课题(No.2013ZX09103003-001)
国家自然科学基金资助项目(No.81270362)
