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目的 :建立表达大鼠NMDA(N methyl D aspartate)受体NR1a与NR2A亚单位重组体的细胞模型 ,研究其生物学及药理学特性。方法 :利用脂质体转染方法将大鼠NR1a与NR2A亚单位重组体共转染到HEK 2 93细胞中 ,在细胞培养 4 8～ 72h后 ,利用膜片钳全细胞记录技术 ,观察和分析L 谷氨酸诱发的NMDA受体电流。结果 :向转染后的HEK 2 93细胞表面喷射NMDA受体激动剂L 谷氨酸可在 38%细胞上诱发出瞬时内向电流 ,该电流可被NMDA受体选择性拮抗剂D AP5 (5 0 μmol/L)完全阻断。L 谷氨酸诱发的电流具有快速失敏的动力学特征和内向整流特性 ,衰减时间常数 (τ值 )为 (5 3± 9)ms(n =2 0 )。L 谷氨酸诱发电流的EC50 值为 (8.5± 0 .5 ) μmol/L。 结论 :成功地将NR1a与NR2A亚单位转染入HEK 2 93细胞并形成NMDA受体的功能性表达 ,为进一步深入研究NMDA受体不同亚单位组成与其功能的关系及其药理学特性奠定了基础。
OBJECTIVE: To establish a cell model expressing rat NR1a and NR2A subunits of NMDA (N methyl D aspartate) receptor and to study its biological and pharmacological properties. Methods: Rat NR1a and NR2A subunit recombinants were co-transfected into HEK2 93 cells by lipofection method. After cell culture for 48-72 hours, whole-cell patch clamp recording technique was used to observe and analyze L-glutamate-induced NMDA receptor currents. RESULTS: The injection of NMDA receptor agonist L-glutamate on the surface of transfected HEK 2 93 cells induced an immediate inward current on 38% of the cells, which was blocked by NMDA receptor selective antagonist D AP5 (50 μmol / L) completely blocked. The L-glutamic acid-induced current has the kinetic characteristics of fast desensitization and the inward rectification characteristics, with a decay time constant of (5 3 ± 9) ms (n = 20). The EC50 value of L-glutamate induced current was (8.5 ± 0.5) μmol / L. CONCLUSION: The successful expression of NR1a and NR2A subunits into HEK2 93 cells and the formation of functional expression of NMDA receptors lay the foundation for further study of the relationship between different subunits of NMDA receptors and their pharmacological properties .