蛋白激酶B受体在大鼠脑星形胶质细胞的表达及其磷酸化

目的:研究大鼠脑星形胶质细胞蛋白激酶B受体(TrkB)的表达及其信号转导通路。方法:用生后2~3dSD大鼠,在无菌条件下取脑,制备细胞悬液,以胶质纤维酸性蛋白(GFAP)鉴定星形胶质细胞;以RT-PCR法研究星形胶质细胞TrkB、ERK1、ERK2 mRNA表达,用蛋白印迹和免疫细胞化学法研究星形胶质细胞TrkB、ERK1、ERK2蛋白表达。用蛋白印迹法检测脑源性神经营养因子(BDNF)作用后的星形胶质细胞p-TrkB。结果:星形胶质细胞的纯度达95%以上,RT-PCR结果显示星形胶质细胞表达TrkB、ERK1、ERK2 mRNA,蛋白印迹及免疫细胞化学法显示星形胶质细胞表达TrkB、ERK1、ERK2蛋白。BDNF作用1h后TrkB磷酸化,K252a可阻止TrkB磷酸化。结论:培养的大鼠星形胶质细胞表达TrkB、ERK1、ERK2;BDNF可使TrkB磷酸化,TrkB与星形胶质细胞信号转导有关。

Expression and phosphorylation of Trk B in cultured astrocytes of rat brain

Objective: To study the expression of Trk B and its signal transduction in the cultured astrocytes of rat brain. Methods: The brains were obtained from 2-3 days postnatal SD rats under sterile condition. Cell suspension was prepared by trypsin digestion and the purity of astrocyte was identified using GFAP immunocytochemistry. The expression of Trk B, ERK1 and ERK2 mRNA was determined by RT-PCR assay in the cultured astrocytes; the expression of Trk B, ERK1 and ERK2 protein was detected using Western blotting analysis and immunocytochemistry.After the treatment of BDNF for 1 h, phosphorylated Trk B was examined by Western blot. Results: The purity of astrocyte was more than 95%. The RT-PCR analysis showed that the astrocytes expressed the Trk B, ERK1 and ERK2 mRNA; western blotting and immunocytochemical assay demonstrated that the astrocytes expressed the Trk B, ERK1 and ERK2 protein. After the treatment of BDNF for 1 h, Trk B were phosphorylated. Conclusion: These results indicate that the cultured rat brain astrocytes express the trkB, ERK1 and ERK2; Trk B was phosphorylated by BDNF and it may be related with signal transduction of BDNF in astrocytes.