Treatment of neuropathic pain by transfection of mutant NMDA receptor gene to the spinal cord of the rat

作为初步研究，我们计划研究突变型NMDA受体表达对阿片类药物耐受性的影响。将NR1N端616位密码子从AAC突变为CGC的N616R CDNA连接到pcDNA3.1载体上。将神经母细胞瘤×神经胶质瘤(NG108-15)杂交瘤细胞在含10%胎牛血清的Dulbecco‘s改良Eagle’s培养液中培养成单层细胞。用D-Pen2，D-Pen5-脑啡肽(DPDPE)刺激细胞4~12h。然后，用含有10μM Forsklin和500μM 1-甲基-3-异丁基黄嘌呤的培养液刺激细胞10分钟。用酶免疫法测定cAMP的生成。到目前为止，DPDPE治疗没有引起Forsklin刺激的cAMP的高生成，我们计划将其作为阿片类药物耐受性的衡量标准。

As a preliminary study, we planed to study the effects of mutant NMDA receptor expression on opioid tolerance of cultured cells.N616R CDNA, 616th codon from N-terminal of NR1 cDNA, which is one of a NMDA subunit cDNA, has been mutated from AAC to CGC, was ligated into pcDNA3.1 vector. NR2A cDNA, which is a cDNA of another NMDA receptor subunit, was also ligated into pcDNA3.1 vector.Neuroblastoma x glioma(NG108-15) hybrid cells were groum as monolayers in Dulbecco's modified Eagle's medium, containing 10 % fetal bovine serum. The cells were treated with control medium or medium containing delta-selective agonist (D-Pen2, D-Pen5)-enkephalin (DPDPE) for 4 to 12 hours. Thereafter, the cells were challenged with medium containing 10 μM forskolin and 500 μM 1-methyl-3-isobutylxanthine for 10 minutes. Production of cAMP was measured by enzymeimmunoassay. So far, DPDPE treatment did not cause forskolin stimulaeted hyper production of CAMP, which we planed to use as a measure for the opioid tolerance.