NMDA受体在爪蟾卵母细胞和HEK-293细胞表达体系中的建立

Methods to establish Xenopus laevis oocytes and HEK-293 cells expressing NMDA receptors

目的:建立特异性表达NMDA受体(NMDAR)NR2AR、NR2BR亚型的爪蟾卵母细胞和HEK-293细胞模型,选出合适的表达体系模型,为研究药物对NMDAR介导电流的影响奠定基础。方法:分别采用显微注射法和脂质体转染法将NMDAR表达在爪蟾卵母细胞和HEK-293细胞上,利用全细胞膜片钳技术,记录NMDAR亚型NR2AR、NR2BR介导的电流。结果:在建立的爪蟾卵母表达体系中,NMDAR基因注射成功率为100%,且能检测到NMDAR亚型NR2AR和NR2BR介导的内向电流,而在HEK-293表达体系中,转染成功率为(6.7±1.39)%,未检测到NMDAR介导的电流。结论:成功建立爪蟾卵母细胞NMDAR表达体系,可作为研究药物对爪蟾卵母细胞表达体系上NMDAR电流影响的合适的细胞表达体系模型。在HEK-293表达体系上未检测到相关电流,可能与其转染后细胞活性大大降低、脂质体转染效率低等因素有关。

Objective：To establish Xenopus laevis oocytes and HEK-293 cells models expressing N-methylD-aspartate receptors（NMDAR）comprising NR2 AR/NR2 AR subunits.Methods：Microinjection and liposome transfection were performed respectively to establish Xenopus laevis oocytes and HEK-293 cells expressing NMDAR.Currents induced by NMDAR subunits NR2 AR and NR2 BR were recorded by whole-cell patch-clamp.Results：NMDAR subtypes NR2 AR and NR2 BR-mediated whole cell currents were detected.The success rate of NMDAR gene injection was 100%in Xenopus laevis oocytes,whereas NMDAR mediated currents were not detected in the HEK-293 cells,and the transfection success rate was（6.7±1.39）%.Conclusion：NMDAR was expressed in Xenopus laevis oocytes,providing a cell model to investigate the effects of drugs on the current of NMDAR.However,we failed to examine the currents in HEK-293 expression system,which might be related to low transfection efficiency.