摘要目的 评价多次鞘内注射cAMP反应元件结合蛋白(CREB)反义寡核苷酸对神经病理性痛小鼠脊髓含2A亚基的NMDA受体(NR2A)表达的影响.方法 鞘内置管成功的C57BL/6雄性小鼠40只,体重20 ～ 25 g,采用随机数字表法,将其随机分为4组(n=10):生理盐水组(NS组)、CREB同义寡核苷酸组(S组)、CREB错义寡核苷酸组(M组)和CREB反义寡核苷酸组(A组).采用结扎坐骨神经的方法制备小鼠神经病理性痛模型,结扎坐骨神经后第1～6天,4组分别鞘内注射生理盐水5μl、同义寡核苷酸5 μg/5μl、错义寡核苷酸5μg/5μl、反义寡核苷酸5μg/5μl,1次/d.于术前1d、造模后1、3、5、7d时测定小鼠结扎侧足底的机械缩足阈值(PWMT)和热辐射刺激潜伏期(PWTL).于结扎坐骨神经后7、14 d时各取5只小鼠断头处死,取L3～5脊髓,采用Western blot和RT-PCR法测定NR2A的表达水平.结果 与基础值相比,A组小鼠结扎坐骨神经1～7d时PWMT和PWTL差异无统计学意义(P＞0.05),NS组、S组和M组PWMT和PWTL降低(P＜0.05).与NS组、S组和M组相比,A组小鼠结扎坐骨神经后7、14 d时脊髓NR2A mRNA和蛋白表达下调(P＜0.05).与结扎坐骨神经后7d时相比,4组结扎坐骨神经后14 d时小鼠脊髓NR2A mRNA和蛋白表达上调(P＜0.05).结论 在神经病理性疼痛形成阶段多次鞘内注射CREB反义寡核苷酸可减轻神经病理性痛,其机制可能与抑制脊髓NR2A表达有关.

abstractsObjective To investigate the effects of repeated intrathecal cyclic AMP response elementbinding protein (CREB) antisense oligodeoxynucleotide (ODN) on the expression of NR2A in spinal cord in mice with neuropathic pain produced by chronic constrictive injury of the sciatic nerve (CCI).Methods Forty C57BL/6 male mice in which intrathecal catheter was successfully implanted were randomly divided into 4 groups ( n =10 each):normal saline group (group NS),CREB sense ODN group (group S),CREB missense ODN group (group M),and CREB antisense ODN group (group A).In groups NS,S,M and A,normal saline 5μl,sense ODN 5 μg/5 μl,missense ODN 5 μg/5 μl and antisense ODN 5 μg/5 μl were injected intrathecally once a day for 6 days,starting from the 1st day after CCI,respectively.Paw withdrawal mechanical threshold (PWMT) and paw withdrawal thermal latency (PWTL) were measured on day 1 before CCI and on day 1,3,5 and 7 after CCI.Five mice from each group were sacrificed on day 7 and 14 after CCI and the lumbar segment of the spinal cord (L3-5 )was removed for determination of NR2A expression using Western blot and RT-PCR.Results Compared with the baseline value,no significant change was found in PWMT and PWTL on day 1-7 after CCI in group A ( P ＞0.05),while PWMT and PWTL were significantly decreased on day 1-7 after CCI in groups NS,S and M (P ＜0.05).Compared with groups NS,S and M,the expression of NR2A mRNA and protein was significantly downregulated on day 7 and 14 after CCI in group A ( P ＜ 0.05).The expression of NR2A mRNA and protein was significantly up-regulated on day 14 after CCI compared with that on day 7 after CCI in all the groups.Conclusion Intrathecal CREB antisense ODN during the development of neuropathic pain can attenuate neuropathic pain and inhibition of the expression of NR2A in mouse spinal cord may be involved in the mechanism.