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

突变型NMDA受体基因转染大鼠脊髓治疗神经病理性疼痛

• 批准号: 12671482
• 负责人: SUZUKI Nobuaki
• 金额: $ 2.11万
• 依托单位: 宮崎医科大学
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12671482/
• 关键词: NMDA receptor; delta opioid receptor; CAMP; Mutant NMDA receptor; 変異型NMAD受容体遺伝子; デルタ受容体

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.

作为一项初步研究，我们计划研究突变型 NMDA 受体表达对培养细胞的阿片类药物耐受性的影响。 N616R cDNA，NR1 cDNA N 端第 616 个密码子，是 NMDA 亚基 cDNA 之一，已从 AAC 突变为 CGC，连接到 pcDNA3.1 载体中。 NR2A cDNA，它是另一个NMDA受体亚基的cDNA，也被连接到pcDNA3.1载体中。神经母细胞瘤x神经胶质瘤(NG108-15)杂交细胞在含有10％胎牛血清的Dulbecco改良Eagle培养基中形成单层。将细胞用对照培养基或含有δ-选择性激动剂(D-Pen2、D-Pen5)-脑啡肽(DPDPE)的培养基处理4至12小时。此后，用含有10μM毛喉素和500μM 1-甲基-3-异丁基黄嘌呤的培养基攻击细胞10分钟。通过酶免疫测定法测量cAMP的产生。到目前为止，DPDPE 治疗并未导致毛喉素刺激 CAMP 的过度产生，我们计划将其用作阿片类药物耐受性的衡量标准。