鱼藤酮诱导BV2小胶质细胞半胱氨酰白三烯受体1表达变化  

Rotenone-induced changes of cysteinyl leukotriene receptor 1 expression in BV2 microglial cells

作　　者：骆江云[1] 张壮[1] 余舒莹[1] 赵冰[1] 赵春贞[1] 王欣欣[1] 方三华[1] 张纬萍[1] 张丽慧[2] 魏尔清[1] 卢韵碧[1] 

机构地区：[1]浙江大学医学院药理学系,浙江杭州310058 [2]杭州师范大学医学神经生物学市级重点实验室基础医学部药理学教研室,浙江杭州310036

出　　处：《浙江大学学报（医学版）》2011年第2期131-138,共8页Journal of Zhejiang University(Medical Sciences)

基　　金：国家自然科学基金资助项目(30700150;30873053;81072618);浙江省自然科学基金资助项目(Y2090069);浙江省科技厅钱江人才项目(2010R10055);中央高校基本科研业务费专项资金

摘　　要：目的:制备和鉴定半胱氨酰白三烯受体1(CysLT1受体)抗体,以此观察鱼藤酮诱导BV2小胶质细胞损伤过程中CysLT1受体表达变化。方法:以KLH偶联的CysLT1受体多肽(小鼠)免疫家兔制备多克隆抗体,用ELISA法测定抗体效价,以抗原阻断法测定抗体敏感性及特异性。用新制备的抗体以Western blotting检测鱼藤酮(0.01～1μmol/L,作用24 h)损伤BV2细胞过程中,BV2细胞上CysLT1受体的表达变化,并以RT-PCR检测验证CysLT1受体mRNA表达水平,同时以免疫组化观察CysLT1受体的亚细胞分布变化。结果:ELISA测定显示,制备的兔抗小鼠CysLT1受体抗体的效价大于1/32728,对CysLT1受体的特异性强,对CysLT2受体和GPR17基本无交叉反应。Western blotting和RT-PCR显示,鱼藤酮作用BV2细胞24 h剂量依赖性诱导CysLT1受体mRNA及蛋白水平表达升高;免疫组化显示在鱼藤酮诱导BV2细胞损伤过程中,随鱼藤酮剂量增加,CysLT1受体发生核移位。结论:制备的CysLT1受体多克隆抗体具有高效价和高特异性,能满足Western blotting和免疫组化检测的要求;CysLT1受体参与鱼藤酮诱导的BV2细胞损伤。Objective:To prepare and identify a polyclonal antibody(pAb) against(mouse) cysteinyl leukotriene receptor 1(CysLT1) and to investigate the changes of CysLT1 receptor expression in BV2 microglial cells after rotenone treatment. Methods:Rabbits were immunized with KLH-coupled CysLT1 peptide to prepare the pAb.The titer of the pAb in rabbit plasma was detected by ELISA method,and the specificity of the pAb was tested by antigen blockade.After BV2 cells were treated with rotenone(0.01-1 μmol/L) for 24 h,the expression of CysLT1 was determined by immunostaining,Western blotting and RT-PCR. Results:The pAb showed a titer of 1/32728,and was not cross-reacted with antigens of CysLT2 receptor and GPR17.Immunostaining,Western blotting and RT-PCR analysis showed the expression of CysLT1 receptor in BV2 microglia.Rotenone at 1μmol/L significantly induced an increased expression of CysLT1 receptor. Conclusion:The prepared CysLT1 receptor polyclonal antibody has a high titer and high specificity to meet testing requirements of Western blotting and immunostaining;CysLT1 is associated with rotenone-induced injury of BV2 microglial cells.

关 键 词：半胱氨酸 受体 白三烯 抗体 鱼藤酮/药理学 半胱氨酰白三烯受体 多克隆抗体 BV2小胶质细胞 

分 类 号：R741[医药卫生—神经病学与精神病学]