能合成和分泌GABA的永生化星形胶质细胞的构建

Construction of genetically immortalized rat astrocytes to produce and release GABA

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 conctruct genetically Engineered cell lines to produce and release GABA through the immortalized rat astrocytes. Methods By immortalized rat astrocytes as cell carriers, we constructed cell lines with the GABA-synthesizing enzyme GAD65 cDNA. The GABA-producing cells expressed GAD65 with immunohisochemistry and Western-blot; The content of GABA was deteced with Capillary Electropherograms; GABA current was recorded in cell lines using whole-cell voltage clamp technique. Results Compared with the controls, the content of GAD65 in GABA-producing cells increased obviously by immunohisochemistry and Western-Blot. By Capillary electropherograms, the content and secretion of GABA of the GABA-producing cells was markly increased（P〈0. 05）; GABA current was recorded in the GABA-producing cells by the whole cell voltage clamp technique. Conclusions Using the immortalized rat astrocytes as cell carriers, the GABA-producing cells were engineered successfully, and the cell lines can produce and release GABA.