Tan P-H, Yang L-C, Shih H-C, Lan K-C, Cheng J-T
Department of Biological Sciences, National Sun Yat-Sen University, Kaohsiung, Taiwan, ROC.
Gene Ther. 2005 Jan;12(1):59-66. doi: 10.1038/sj.gt.3302376.
N-methyl-D-aspartate (NMDA) receptor activation, at the level of the spinal cord, has been shown to play an important role in the facilitation of nociception in several animal models. However, the use of NMDA antagonists as analgesics is limited by serious side effects due to nonselective effects among the NMDA receptor subtypes. Recent discoveries revealed that the transfection of small interfering RNAs (siRNAs) into animal cells resulted in the potent, long-lasting, post-transcriptional silencing of specific genes. Thus, we investigated the effect of intrathecal (i.t.) injection of siRNAs targeting NMDA-R2B receptor subunit protein (NR2B) receptors, a subunit of NMDA receptor, for the modulation of pain. The results indicate that the use of siRNA targeting the NR2B subunit not only decreased the expression of NR2B mRNA and its associated protein, as demonstrated by real-time PCR and Western blotting, but also abolished formalin-induced pain behaviors in rat model. The peak effect occurred on day 3 for mRNA and day 7 for its protein, following i.t. injection of 5 microg of siRNA-NR2B. These data prove the feasibility of i.t. siRNAs in the investigation of functional gene expression in the context of whole animal behavior for the management of chronic pain.
在几种动物模型中，已表明脊髓水平的N-甲基-D-天冬氨酸（NMDA）受体激活在伤害感受的促进过程中起重要作用。然而，由于NMDA受体亚型之间的非选择性作用，NMDA拮抗剂作为镇痛药的使用受到严重副作用的限制。最近的发现表明，将小干扰RNA（siRNA）转染到动物细胞中会导致特定基因的有效、持久的转录后沉默。因此，我们研究了鞘内（i.t.）注射靶向NMDA-R2B受体亚基蛋白（NR2B）受体（NMDA受体的一个亚基）的siRNA对疼痛调节的影响。结果表明，使用靶向NR2B亚基的siRNA不仅降低了NR2B mRNA及其相关蛋白的表达，这通过实时PCR和蛋白质印迹法得到了证实，而且还消除了大鼠模型中福尔马林诱导的疼痛行为。在鞘内注射5微克siRNA-NR2B后，mRNA的峰值效应出现在第3天，其蛋白质的峰值效应出现在第7天。这些数据证明了鞘内注射siRNA在全动物行为背景下研究功能性基因表达以管理慢性疼痛方面的可行性。
