卵母细胞基因表达系统的应用研究

A Practical Research on Oocytes Gene Expression System

目的 :建立研究神经生理及药理的卵母细胞表达模型 ,并对鸡脑mRNA在卵母细胞上表达的氨基酸类受体进行研究。方法 :采用盐酸胍或异硫氰酸胍法抽提总RNA ,经寡聚脱氧胸苷 (oligo -dT)纤维素柱纯化得mRNA。mRNA微注射到卵母细胞内进行表达 ,以双电极电压钳技术研究表达的受体。结果 :模型能明显表达外源性mRNA表达的受体 ,而阴性对照无表达。在卵母细胞膜上表达的γ -氨基丁酸 (γ -aminobutyricacid ,GABA) ,甘氨酸(Glycine ,Gly)及红藻氨酸 (Kainate ,KA)受体 ,施加GABA ,Gly及KA均能引起一剂量依赖的内向电流 ,相应的拮抗剂可阻断其电流。剂量效应曲线显示GABA及KA诱导电流的半有效浓度 (EC5 0 )分别为 2 .9× 10 -5mol/L及 6 .3× 10 -5mol/L。结论 :卵母细胞可明显表达鸡脑mRNA编码的KA ,Gly及GABA受体 ,其药理特性与天然受体相似 ,是一个良好的神经生理及药理研究模型。

Objective To establish the oocyte model for neurophysiological and neuropharmacological studies, and to investigate GABA, Gly and KA receptors expressed in Xenopus Laevis oocytes.Methods GABA, Gly and KA receptors were investigated with two electrodes voltage-clamp technique after micro injection of mRNA from chicken brain, where mRNA was isolated with guinidine hydrochloride- or guinidinium thiocyanate-phenol chloroform.Results The expressions of GABA, Gly and KA receptors were clear.All GABA-, Gly- and KA-induced currents were inward concentration-dependent currents. The half-excitation concentration of GABA- and KA-induced currents was 2.9×10 -5 mol/L and 6.3×10 -5 mol/L repectively.Conclusion Oocytes may be a good model for neurophysiolodical and neuropharmacologicoal studies, for GABA, Gly and KA receptors are clearly expressed.