鲟鱼外周血淋巴细胞的分离及最佳体外增殖性反应条件  

Isolation of Peripheral Blood Lymphocytes of Sturgeon and the Optimal Proliferate Response Condition

作　　者：董颖[1] 胡红霞[1] 田照辉[1] 王巍[1] 东天[1] DONG Ying;HU Hong-xia;TIAN Zhao-hui;WANG Wei;DONG Tian(Beijing Fisheries Research Institute and National Engineering Research Center for Freshwater Fisheries, Beijing Key Laboratory of Fishery Biotechnology, Beijing 100068)

机构地区：[1]北京市水产科学研究所暨国家淡水渔业工程技术研究中心渔业生物技术北京市重点实验室,北京100068

出　　处：《生物技术通报》2018年第3期150-155,共6页Biotechnology Bulletin

基　　金：科技部支撑计划(2015BAD25B00);现代农业产业技术体系北京市鲟鱼;鲑鳟鱼创新团队(BAIC08-2018);北京市优秀人才培养资助(2014000020060G183);北京市农林科学院青年科研基金(QNJJ201506);冷水性鱼类科技创新团队(JNKST201611)

摘　　要：以小体鲟(Acipenser ruthenus)为研究对象,应用不同浓度的淋巴细胞分离液对鲟鱼外周血淋巴细胞进行分离并探讨其增殖性反应的最佳条件。分别以植物血凝素(PHA)、刀豆蛋白A(Con A)和脂多糖(LPS)作为淋巴细胞增殖丝裂原,采用L25(56)五因素五水平正交试验设计,用Enhanced Cell Counting Kit-8法(增强型CCK-8或WST-8)对培养时间、培养温度、细胞密度、胎牛血清浓度及丝裂原浓度等因素进行探索性研究。结果表明:70%的Percoll液(比重为1.092 g/m L)作为淋巴细胞分离液的分离效果最佳。鲟鱼最佳外周血淋巴细胞体外增殖反应条件为:3.625×10~6初始细胞量、20μg/m L的PHA或50μg/m L的Con A或10μg/m L的LPS作为丝裂原、10%-20%胎牛血清(FBS)、20-25℃培养2 d。In this study,sterlet（Acipenser ruthenus）was chosen as the model experimental material,different concentrations oflymphocyte separation solution were used to isolate the peripheral blood lymphocytes of sturgeon and study their optimal proliferate responsecondition. Phytohemagglutinin（PHA）,concanavalin A（Con A）and lipopolysaccharide（LPS）were utilized as lymphocyte proliferationmitogen respectively. According to five factors and five levels orthogonal experimental design L_（25）（5~6）,the conditions of sturgeons peripheralblood lymphocytes proliferate response were optimized using Enhanced Cell Counting Kit-8（enhanced CCK-8 or WST-8）. Five factors wereselected to explore the optimal response condition,including culture time,culture temperature,cell concentration,fetal bovine serum（FBS）concentration,and mitogen concentration. The results showed that 70% Percoll（1.092 g/m L）used as the sturgeon lymphocyte separationsolution had the best separation effect. The optimal conditions for proliferation were 3.625×10~6 initial cell concentration,20 μg/m L PHA or 50μg/m L Con A or 10 μg/m L LPS,10%-20% FBS,20-25℃,and culturing for 2 days.

关 键 词：鲟鱼 外周血淋巴细胞 淋巴细胞分离 增殖性反应 植物血凝素 刀豆蛋白A 脂多糖 CCK-8 

分 类 号：S917.4[农业科学—水产科学]