受体相互作用蛋白140在小鼠原代小胶质细胞中的表达分析

Gene expression of receptor-interacting protein140 in mouse microglia cells

目的:分离、纯化和原代培养小鼠的小胶质细胞并检测该细胞中受体相互作用蛋白140(RIP140)的表达模式。方法:以McCarthy等的经典培养方法为基础进行小鼠小胶质细胞的分离,以不换液营养缺失法、机械振摇法纯化和培养小鼠小胶质细胞;用CD11b(MAC-1)对分离的小鼠小胶质细胞进行鉴定;采用实时-聚合酶链反应和western免疫印迹法检测RIP140mRNA和蛋白质在小鼠原代小胶质细胞中的表达模式;用免疫细胞化学确定RIP140在小胶质细胞中的定位表达模式。结果:成功对小鼠小胶质细胞进行了分离、纯化和培养;mRNA和蛋白表达分析显示,RIP140在小鼠原代小胶质细胞中确有表达,免疫组化结果提示RIP140主要在小鼠小胶质细胞的细胞核中表达。结论:利用McCarthy经典改良方法可成功分离纯化出高纯度小鼠原代小胶质细胞;小鼠原代小胶质细胞确实表达RIP140,以细胞核表达为主。

Objective：To isolate,purify and primary culture the mouse microglia cell and to detect the expression of receptor-interacting protein140 （RIP140）in microglia cells.Methods：The isolation of microglia cell was based on the classic method of McCarthy,the purification and culture were modified by nutrition deprivation and mechanical shaking.CD11b （MAC-1）antibody was used to identify the primary microglia with immunohistology.Real time-polymerase chain reaction,western blotting and immunohistology were used to detect the gene expression and localization of RIP140 in mouse microglia cells.Results：The primary culture of microglia was successfully performed.RIP140 was expressed in microglia cells and localized in its nuclear.Conclusion：The pure mouse microglia cell can be isolated by using the upgrade classic method of McCarthy.RIP140 is clearly expressed in the primary microglia cells.