小鼠大脑皮质星形胶质细胞的培养、鉴定及其TRPC1-7 mRNA的表达

The Culture and Expression of TRPC1-7 mRNA in Astrocytes from Cerebral Cortex of Mice

目的建立有效的星形胶质细胞体外培养体系及检测TRPC1-7 mRNA在其中的表达,为体外研究星形胶质细胞TRPC通道与神经系统疾病的关系奠定基础。方法本实验于无菌条件下取新生小鼠的大脑皮质,经胰酶消化及机械吹打分离细胞,差速贴壁法处理以去除成纤维细胞,用含15%胎牛血清的DF12培养液培养。之后摇床振荡去除小胶质细胞,并以GFAP对星形胶质细胞进行鉴定,PCR检测其中TRPC1-7 mRNA的表达。结果分离培养的细胞有典型的星形胶质细胞形态,并表达GFAP,纯化率95%,且其中TRPC1-7 mRNA均有表达。结论本实验方法可获得符合体外研究要求的星形胶质细胞并有TRPC1-7 mRNA表达,为体外研究星形胶质细胞TRPC通道与神经系统疾病的关系奠定基础。

Objective To establish an effective method of cultured cerebral cortical astrocyte in mice,and detect the expression of TRPC1-7 mRNA in cultured mice astrocytes.Methods In this experiment,the astrocytes were isolated from the cerebral cortex of newborn mice by trypsin digestion and mechanical blow under aseptic condition,and the fibroblasts were removed by differential adherent method.The cells were cultured in DF12 medium containing 15%fetal bovine serum.And then,it purified by shaking the culture flasks to remove microglial cells in shaker at 37℃,and identified GFAP by immunofluorescence staining.Moreover,the expression of TRPC1-7 mRNA was determined via real-time quantitative polymerase chain reaction.Results The purity of GFAP-positive cells reached 95%,and TRPC1-7 mRNA were also expressed in cells.Conclusion This experimental method can obtain astrocytes that meet the requirements of in vitro research and have TRPC1-7 mRNA expression,which lays a foundation for the study of the relationship between astrocyte TRPC channel and nervous system diseases in vitro.