单核细胞的原代分离方法优化及其炎症激活模型的构建  

Optimization of primary isolating monocytes method and establishing of monocytes inflammatory activation model

作　　者：田敏 胡俊[2] 杨国华[2] 谢明水[3] 黄宏耀[3] TIAN Min;HU Jun;YANG Guohua;XIE Mingshui;HUANG Hongyao(Graduate Education Base of Hubei Medical College,Jinzhou Medical University,Suizhou 441300,Hubei,China;Basic Medical Experimental Teaching Center,Wuhan University,Wuhan 430000,Hubei,China;Departmert of Laboratory,Suizhou Central Hospital,Hubei Medical College,Suizhou 441300,Hubei,China)

机构地区：[1]锦州医科大学湖北医药学院研究生培养基地,湖北随州441300 [2]武汉大学基础医学实验教学中心,湖北武汉430000 [3]湖北医药学院附属随州中心医院检验科,湖北随州441300

出　　处：《生物资源》2020年第2期221-227,共7页Biotic Resources

基　　金：湖北省卫生计生委面上项目(WJ2017M003)。

摘　　要：为了得到高纯度高得率的单核细胞(monocyte)并分析其在血栓发生中的作用,本研究利用10位健康人的血样分离出单个核细胞(peripheral blood mononuclear cell,PBMC),并通过免疫磁珠分选来获得单核细胞,测定PBMC与单核细胞的得率。通过制备血浆中的高丰度蛋白β2GPI,并与其抗体构成抗原抗体复合物来刺激细胞培养,通过荧光定量PCR检测细胞培养基中组织因子TF和TNF-α的含量,构建单核细胞炎症激活模型。实验结果显示单核细胞的得率较高,纯度和活性较好,且β2GPI纯化效果好。荧光定量PCR结果显示,与对照组相比,添加β2GPI抗原抗体复合物的实验组TF与TNF-αmRNA表达量明显增多。本实验表明纯化的单核细胞被充分激活,在细胞水平上为研究机体炎症反应和血栓形成的分子机理提供了炎症模型。In order to obtain high-purity and high-yield monocytes and analyze their role in thrombosis,this study used 10 healthy human blood samples to isolate PBMC,to obtain monocytes by immunomagnetic bead sorting,and to determine the yield of PBMC and monocytes. By preparing a high abundant protein β2 GPI from plasma,it was combined with its antibody to form an antigen-antibody complex to stimulate cells,and the content of tissue factor TF and TNF-α in cell culture medium were detected by real-time PCR to construct mononuclear cell inflammation activation model. The experimental results showed that the yield of monocytes was higher,and the purity and activity were better,and the purification effect of β2 GPI was also improved. The results of real-time PCR showed that compared with the control group,the expression levels of mRNA of TF and TNF-α in the experimental group with the antigen-antibody complex significantly increased. This experiment demonstrates that purified monocytes are fully activated and provide an inflammatory model at the cellular level for studying the molecular mechanisms of inflammatory response to and thrombosis.

关 键 词：单核细胞 Β2糖蛋白I 组织因子 肿瘤坏死因子⁃α 单核细胞激活 炎症模型 

分 类 号：R34[医药卫生—基础医学]