脊髓背角NMDA受体NR2B亚基对舒芬太尼耐受形成的影响研究

Effect of NMDA receptor NR2B subunit on the tolerance of sufentanil in spinal dorsal horn

目的研究脊髓背角NMDA受体NR2B亚基对舒芬太尼耐受形成的影响。方法 32只成年雄性小鼠随机分为2组,每组16只,A组小鼠采用逐日递增法皮下注射生理盐水;B组小鼠经皮下注射舒芬太尼建立舒芬太尼耐受小鼠模型,并通过检测小鼠机械缩足反射阈值及热缩足反射潜伏期进行验证,同时合成并筛选对NR2B基因抑制效果较好的siRNA。对舒芬太尼耐受小鼠鞘内注射siRNANR2B后,检测舒芬太尼耐受小鼠机械缩足反射阈值及热缩足反射潜伏期,同时通过RT-PCR、Western blot技术分析舒芬太尼耐受小鼠NR2B亚基表达水平变化。结果与A组相比,B组小鼠的机械缩足反射阈值及热缩足反射阈值检测明显下降(P<0.05)。通过RT-PCR及Western blot实验发现,舒芬太尼耐受小鼠NR2B亚基表达水平上升(P<0.05);将siRNA-1经鞘内注射舒芬太尼耐受小鼠后,siRNA-1可显著降低舒芬太尼耐受小鼠脊髓背角组织中NR2B的mRNA及蛋白水平(P<0.05)。通过RT-PCR及Western blot实验发现,siRNA-1对脊髓背角细胞的NR2B的mRNA及蛋白抑制效果较好(P<0.05)。舒芬太尼耐受小鼠转染siRNA后,舒芬太尼耐受小鼠的机械缩足反射阈值及热缩足反射阈值显著高于B组(P<0.05)。结论脊髓背角NMDA受体NR2B亚基与舒芬太尼耐受形成密切相关,减少体内NR2B亚基水平可能增强舒芬太尼的镇痛效果。

Objective To study the effect of NMDA receptor NR2B subunit on the formation of sufentanil tolerance in spinal dorsal horn.Methods 32 adult male mice were randomly divided into 2 groups,16 in each group.Group A mice were injected with normal saline subcutaneously daily,while group B mice were subcutaneously injected with sufentanil to establish sufentanil tolerance.The mouse model was validated by detecting the mechanical withdrawl threshold and paw withdrawal thermal latency of mice.At the same time,siRNAs with better inhibition effect on NR2B gene were synthesized and screened.After intrathecal injection of siRNA-NR2B in sufentanil-resistant mice,the mechanical withdrawl threshold and paw withdrawal thermal latency of sufentanil-resistant mice were detected.The expression levels of NR2B subunits in sufentanil-resistant mice were analyzed by RT-PCR and Western blot.Results Compared to group A,the mechanical withdrawl threshold and paw withdrawal thermal latency of mice in group B was significantly decreased(P<0.05).The expression of NR2B subunit in sufentanil-tolerant mice increased(P<0.05).After intrathecal injection of siRNA-1 into sufentanil-tolerant mice,the expression of NR2B mRNA and protein in spinal dorsal horn of sufentanil-tolerant mice decreased significantly(P<0.05).RT-PCR and Western blot showed that siRNA-1 inhibited the mRNA and protein of NR2B in spinal dorsal horn cells(P<0.05).The mechanical withdrawl threshold and paw withdrawal thermal latency of sufentanil tolerance mice with siRNA-1 intrathecal injection were significantly higher than those of B group(P<0.05).Conclusion The NMDA receptor NR2B subunit of spinal dorsal horn is closely related to the formation of sufentanil tolerance.Decreasing the level of NR2B subunit in vivo may enhance the analgesic effect of sufentanil.