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目的构建能合成和分泌GABA的永生化星形胶质细胞。方法在原代培养的大鼠星形胶质细胞内转入猴肾病毒40大T抗原基因(si mian virus40large T antigen gene,SV40Tag)使其永生化(这一部分由同济医院麻醉科田玉科教授实验室完成),并在这些细胞内转入含谷氨酸脱羧酶65亚型(glutamate de-carboxylase65,GAD65)目的基因质粒,对照组转染含β-gal(β-半乳糖苷酶,对照质粒)质粒;应用免疫组化及Western-blot方法检测转染后细胞内GAD65的表达水平;应用毛细管电泳法检测这些细胞内外的氨基丁酸(GABA)含量;用全细胞膜片钳记录构建细胞的GABA电流。结果与转染β-gal的对照组比较,转染GAD65的实验组细胞在具备胶质细胞特性之外,能稳定表达GAD65,细胞内的GAD65含量明显增加;同时细胞内的GABA含量明显高于对照组;实验组细胞外液GABA的浓度明显高于对照组;实验组和对照组均能记录到GABA电流,说明构建细胞功能完善。结论永生化的星形胶质细胞中转入GAD65质粒后具备胶质细胞特性,构建细胞能稳定表达GAD65,并且能合成和分泌GABA。
Objective To construct immortalized astrocytes capable of synthesizing and secreting GABA. Methods The primary cultured rat astrocytes were transfected with SV40Tag to make it immortalized (this part was completed by the laboratory of anesthesia Hiroyuki Kodake, Tongji Hospital) ) And transfected into these cells with the gene of glutamate de-carboxylase 65 (GAD65), and the control group transfected with the plasmid containing β-gal (β-galactosidase, control plasmid) The expression of GAD65 was detected by immunohistochemistry and Western-blot. The content of GABA in these cells was detected by capillary electrophoresis. The GABA currents of the cells were recorded by whole-cell patch-clamp. Results Compared with the control group transfected with β-gal, GAD65 transfected GAD65 cells could stably express GAD65 with the characteristics of glial cells. The content of GAD65 in cells increased significantly. The content of GABA in cells was significantly higher than that of GAD65 The concentration of GABA in the extracellular fluid of the experimental group was significantly higher than that of the control group. The GABA currents were recorded both in the experimental group and the control group, indicating that the constructed cells functioned well. CONCLUSION: Immortalized astrocytes have glial characteristics after transfection into GAD65 plasmid. The constructed cells can stably express GAD65 and synthesize and secrete GABA.