抗血小板药物对人骨髓间充质干细胞增殖及分泌功能的影响

Effects of antiplatelet drugs on proliferation and secretion of human bone marrow mesenchymal stem cells

背景:有研究指出氯吡格雷、噻氯匹定可引起粒细胞减少,阿司匹林可抑制内皮祖细胞增殖。目前缺乏这些抗血小板药物会对移植入心肌的骨髓间充质干细胞产生何种作用的报道。目的:探讨氯吡格雷、噻氯匹定和阿司匹林对人骨髓间充质干细胞增殖及分泌功能的影响。设计、时间和地点:细胞学体外观察,于2006-03/12在上海市疾病预防控制中心毒理学实验室完成。材料:第2代人骨髓间充质干细胞由上海第九人民医院组织工程研究中心提供。氯吡格雷、噻氯匹定为杭州赛诺菲圣德拉堡民生制药有限公司产品,批号分别为国药准字J20040006和国药准字H19980186。阿司匹林由拜耳医药有限公司生产,批号为国药准字20050059。方法:骨髓间充质干细胞解冻后,加入含100U/mL青霉素、100mg/L链霉素、10%胎牛血清的低糖DMEM体外扩增培养,传至第6代,分别用不同浓度的氯吡格雷、噻氯匹定、阿司匹林进行干预处理,并设立空白对照。主要观察指标:MTT比色法检测细胞增殖情况,流式细胞仪检测细胞表面抗原表达,ELISA法测定细胞培养液中血管内皮生长因子浓度。结果:反映骨髓间充质干细胞增殖变化的A570值在0.02,0.1,0.4,2,10,40μmol/L氯吡格雷或噻氯匹定作用后均明显高于空白对照(P<0.01);经60,600,2000μmol/L阿司匹林处理后则低于空白对照(P<0.05)。细胞表面抗原CD34、CD105、CD106阳性细胞百分率经3种抗血小板药物作用后与空白对照基本相似。与空白对照比较,经0.02μmol/L氯吡格雷及5,2000μmol/L阿司匹林处理后血管内皮生长因子含量无明显变化(P>0.05);40μmol/L氯吡格雷或噻氯匹定作用后血管内皮生长因子含量均显著降低;0.02μmol/L噻氯匹定作用后血管内皮生长因子含量显著升高(P<0.01)。结论:氯吡格雷和噻氯匹定对人骨髓间充质干细胞具有明显的促增殖作用,阿司匹林可抑制其增殖。高浓度氯吡格雷和噻氯匹定能够抑制人骨髓间充质干细胞分泌血管内皮生长因子,低浓度噻氯匹定可促进其分泌。此3种抗血小板药物对人骨髓间充质干细胞的进一步分化无明显影响。

BACKGROUND： Results from clinical trials suggested that clopidogrel and ticlopidine had side effects of granulopenia, and aspirin could inhibit endothelial progenitor cell proliferation. There is no report of effects of these drugs on human bone marrow mesenchymal stem cells （hBMSCs） in stem cell transplantation. OBJECTIVE： To investigate the effects of antiplatelet drugs including clopidogrel, ticlopidine and aspirin on hBMSC proliferation and secretion. DESIGN, TIME AND SETTING： The cytology in vitro observation was performed at the Laboratory of Toxicology, Shanghai Municipal Center for Disease Control and Prevention from March to December 2006. MATERIALS： The second passage of hBMSCs was kindly donated from Shanghai Tissue Engineering Research ＆ Development Center, Shanghai Ninth People＇s Hospital. Clopidogrel （Lot number J20040006） and ticlopidine （Lot number H19980186） were obtained from Hangzhou Sanofi-Synthelabo Minsheng Pharmaceutical CO., Ltd. Aspirin （Lot number 20050059） was obtained from Bayer Vital GmbH. METHODS： The standard culture medium consisted of DMEM-LG, 10% heat-inactivated FBS, 100 U/mL penicillin and 100 μ g/mL streptomycin. After being cultured in vitro expanded out to passage 6, hBMSCs were treated with antiplatelet drugs of different concentrations and compared with control group. MAIN OUTCOME MEASURES： Cell proliferation was assessed by 3- （4, 5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide （MTF） colorimetric assay, level of vascular endothelial growth factor （VEGF） of culture medium was detected by enzyme-linked immunoadsordent assay （ELISA）, and surface antigens of hBMSCs were analyzed by the flow cytometry. RESULTS： A570 values of hBMSCs treated by clopidogrel or ticlopidine of 0.02, 0.1, 0.4, 2, 10, 40 μ mol/L were higher than control group （P 〈 0.01）, while A570 values of aspirin group of 60, 600, 2 000 μ mol/L were lower than control gronp（P 〈 0.05）. Antiplatelet drugs had no obvious effect on cell surface antigens （CD34, CD105, CD106） expressed by hBMSCs. Treated by high dose clopidogrel or ticlopidine （40 μ tool/L）, VEGF level from hMSCs was lower than that of control group（P 〈 0.01）, but VEGF level of low dose （0.02 μ mol/L） ticlopidine group was higher than control group （P 〈 0.01 ） , and there was no significantly difference of VEGF level among low dose clopidogrel group （0.02 μ mol/L）, aspirin group （5, 2 000 μ mol/L）, and control group（P 〉 0.05）. CONCLUSION： Clopidogrel and ticlopidine improve proliferation of hBMSCs, but aspirin inhibits proliferation of hBMSCs. High dose of clopidogrel and ticlopidine suppress VEGF secretion of hBMSCs, while low dose of ticlopidine promote it. Antiplatelet drugs have no obvious effect on hBMSCs differentiation.