高凝状态患者外周血单核细胞促凝血活性及体外干预试验

Procoagulative activity of human peripheral blood monocytes in patients with

high coagulative state and in vitro intervening examination on them Sun Aihong，

Shi Saizhu，Ni Zanming.

Department of Hernatology of Subei People’s Hospital of Jiangsu Province，Yangzhou225001.

【Abstract】 Objective Procoagalative activity（PCA）and plasminogen activity（PA）of human peripheral blood monocytes（HPMC）in patients with high coagulative state such as diabetes mellitus（DM），coronary artery dis_ˉease（CAD）andsolid tumor（ST）and normal persons were studied.At the same time，the effect of injections such as Traditional Chinese Medicine injection of danshen and huangqi on the HPMC in vitro from patients with high coagula_ˉtive state was investigated.Methods HPMCs from these patients and normal persons were sored by density gradient Fieoll-Hypaque and cultured48hours in vitro.PCA and PA from these patients and mormal persoms were examined by assay of chromophorous substrate.The effects of Traditional Chinese Medicine injection of danshen and huangqi on PCA and PA expressing in vitro cultured moncytes were assessed.Results Our results revealed that PCA of HPMC in patients with high coagulative state were higher significantly than those of normal controls.PCA expressing in in vitro cultured monocytes in normal persoms had increasad signifieantly（P<0.05）after stimulated by lipopolysaccharide（LPS），while PA had no effect.PCA were inhibited when injections danshen and huangqi were added to the culture medium at the same time，however，there had been a significantly inhibitory effect on the group of adding injection dan and huangqi to in vitro culture medium.Conclusion There were pronounced higher PCA of patients from DM，CAD and ST was sorted HPMC than that of normal patients.After cultured for48hours in vitro，PCA of sorted HPMC from theses patients was inhibited after having been supplemented with Traditional Chinese Medicine injection of danshen and huangqi.

Key words high coagulative state human peripheral blood monocytes procoagulative activity in vitro in_ˉtervening

近年来研究认为，人外周血单核细胞（HPMC）可产生和（或）结合组织因子（TF）等凝血因子。通过内/外源性凝血途径启动凝血系统表达其促凝血活性（PCA）;而且它也可合成和（或）结合纤溶酶原激活物及抑制物等纤溶组分，参与调节机体的凝血与纤溶平衡 ^［1］。Eggesbq ^［2］ 等研究发现HPMC表达PCA和PA随高密度脂蛋白（HDL）的浓度而变化，HDL浓度低的患者表达PA升高;在细菌脂多糖（LPS）刺激下，PCA和PAI-1明显上升，而t-PA和u-PA均下降。本研究用改良二期发色底物法检测高凝状态患者如糖尿病、冠心病及实体肿瘤患者的HPMC的PCA和PA表达，并用LPS作为刺激剂，丹参、丹参注射液或黄芪注射液作为体外干预药物，以初步了解单核细胞的促凝血活性作用。

1 材料与方法

1.1 主要试剂 （1）淋巴细胞分离液:上海试剂二厂生产。（2）RPMI1640培养液:RPMI164010.4g，谷氨酸2mmol/L，青霉素100IU/ml，链霉素100μg/ml，Na ₂ CO ₃ 2.0g，HEPES476.6mg，配成1000ml。（3）S2222:上海申海生化科技公司生产，2mg/ml以0.85%NaCl溶解。（4）组织凝血活酶:上海通用医疗器械公司生产（上海医科大学华山医院监制），国际敏感指数（ISI）为1.78。冻融，以0.1mol/L Tris缓冲溶液倍比稀释。FVII/FX溶液:参考魏氏 ^［3］ 方法制备，-70℃储存。临用时37℃。（5）细菌脂多糖（LPS）:美国Sigma公司生产。（6）丹参、黄芪注射液:华山医院制剂室提供，3mg/ml。（7）t-PA活性检测试剂盒:上海医科大学分子遗传实验室提供。（8）PAI-1活性检测试剂盒:上海医科大学分子遗传实验室提供。