推拿对慢性疼痛大鼠囊泡膜谷氨酸转运体2表达的影响及其机制研究

The key thorny-issue of treating chronic pain is the unknown mechanism of pain transmission. The spinal-doral-horn is an important node in the pain signals processing conversion. The latest research shows that the VGLUT2 within spinal-doral-horn is the neurotransmitter markers that is closely associated with body nociceptive information transmission. Previous clinic and experiment indicated that the massage has good analgesic effect. So we make the hypothesis that massage has the effect of blocking pain transmission by down-regulating the expression of VGLUT2.. 96 rats were randomly divided into normal group, model group, fake massage group, slow virus injection group and the massage group. The hinder four groups were given L5 spinal nerve ligation; false massage group was given dauting; slow virus injection group was given injecting shRNA recombinant virus which can effectively reduce VGLUT2; while the massage group was given massage. In 7, 17 day of experimental cycle, observed behavior and draw materials, then measured the expression of green fluorescent protein in spinal cord neurons of each group rats by using immunofluorescence histochemical staining method. Detected VGLUT2 protein expression by immune protein imprinting technology. To investigate the mechanism of down-regulation VGLUT2 on the influence of nociceptive information transmission and intervention effect of massage, then provide objective basis for massage analgesia.

慢性疼痛治疗的关键难题是疼痛发生传递的机制不明。脊髓背角是痛觉信息加工转换的重要节点，最新研究表明，脊髓背角内的囊泡膜谷氨酸转运体2（VGLUT2）是与躯体痛觉信息传递密切相关的神经递质标记物。前期临床和实验表明推拿具有较好的镇痛作用。我们假说推拿能通过下调VGLUT2的表达而起到阻断疼痛传递的作用。. 拟将96只大鼠随机分为正常组、手术组、模型组、假推拿组、慢病毒注射组和推拿组，后四组进行L5脊神经结扎，假推拿给予抚摸，慢病毒注射组给予注射能有效降低VGLUT2的shRNA重组慢病毒，推拿组给予推拿手法。实验第7、17d观测行为学及取材，免疫荧光组织化学染色法测各组大鼠脊髓神经元内绿色荧光蛋白的表达；免疫蛋白印迹技术检测VGLUT2蛋白的表达。探讨下调VGLUT2对痛觉信息传递影响的机制及推拿的干预效应，为推拿镇痛提供客观依据。

背景：推拿治疗神经病理性疼痛疗效显著，研究其起效机制具有重要意义。方法：将72只雄性SD大鼠随机分为正常组、假手术组、模型组各16只，以及慢病毒注射组、假推拿组、枢经推拿组各8只。模型组、慢病毒注射组、假推拿组、枢经推拿组建立SNL模型，造模7天后正常组、假手术组、模型组随机选择8只进行行为学检测及取材。从造模后第7天开始干预，对慢性病毒注射组大鼠脊髓内注入使VGLUT2下调的慢病毒颗粒，仅注射1次；对假推拿组大鼠轻轻抚摸背部18min，每天1次，共干预10天；对枢经推拿组大鼠采用按摩推拿手法模拟仪依次刺激双侧环跳、风市、阳陵泉三穴，每法每穴1min，共干预10天，干预结束后进行行为学检测及取材，检测脊髓腰膨大节段中VGLUT2蛋白的表达水平。结果：造模7天后，模型组的体重、热痛觉阈值、机械痛觉阈值、斜板试验角度均低于正常组、假手术组（P＜0.05）；免疫组化及Western blot检测均显示模型组的VGLUT2高于假手术组、正常组（P＜0.05）。干预10天后，枢经推拿组与慢性病毒注射组的体重、热痛觉阈值、机械痛觉阈值、斜板试验角度均高于模型组（P＜0.05）；免疫组化及Western blot检测VGLUT2显示枢经推拿组与慢性病毒注射组均低于模型组（P＜0.05）。结论：枢经推拿可下调VGLUT2的表达，进而调节脊髓内突触终末神经活性物质谷氨酸的释放，抑制疼痛信息传递，可能是枢经推拿镇痛的起效机制之一。