电针"足三里"对脊髓损伤结肠动力障碍大鼠per2基因生物钟效应的研究

Many spinal cord injury (SCI) patients suffer from disfunction of gastric empty and purging the bowels which affect their physical or psychological health and even life-span. Fortunately, electro-acupuncture has been accepted to manage the disfunction of gastrointestinal tract. In addition, it is economical, safe and convenience for patients. However, its mechanism and the accurate tache that electro-acupuncture works are still unclear. Our study concentrate on the bowel movement and colonic slow waves by observation fecal matters, basic electrical rhythm and contratile electrical complex, while learn the rhythmic change in per2mRNA, nNOSmRNA and nNOS of colonic longitudinal muscle myenteric plexus by immunohematology, Immuno-histochemistry double staining method and quantitative real time polymerase chain reaction method before and after electro-acupuncture and Mosapride . So that we can observe their relationship above, estimate the effect after acpuncture and Mosapride and learn the possible mechanism. That will enrich the basic theory of acupuncture modulating the disfunction of gastricintestinal tract and time therapy of acupuncture, provide the evidence of pathogenesis and therapeutic mechanism of disfunction of gastrointestinal tract after spinal cord injury. What's important, it will provide a better choice of acupoints and frequence of electro-acupuncture or combination with mosapride in clinical practice.

用重物坠落法(WD)造成脊髓损伤大鼠模型，以结肠基本电节律、收缩复合机电为指标了解脊髓损伤后大鼠结肠功能状态。采用免疫荧光标记、免疫组化双染色技术、实时荧光聚合酶链反应等技术，观察脊髓损伤后肠功能障碍大鼠per2周期性表达的变化、nNOS及ACh周期性表达变化，三者与结肠周期性节律性收缩、BER的关系；观察电针足三里对脊髓损伤大鼠per2基因和nNOS、ACh两种重要神经递质周期性表达的影响，并与全胃肠动力药莫沙必利比较。为电针足三里调节脊髓损伤后肠功能障碍，为针灸临床开展针灸对胃肠道生物钟调节作用提供理论和实验依据。

背景：肠道运动具有昼夜生物节律，主要是由位于视交叉上核和肠道包括per2在内的生物钟基因调控的。脊髓损伤会导致肠道运动障碍。.目的：评估电针对失中枢支配的脊髓损伤大鼠肠道运动昼夜生物节律及结肠per2基因表达生物钟节律的影响。.方法：控制大鼠生活的昼夜节律，采用重物坠落法制备脊髓损伤大鼠模型。电针组予光照条件下定时电针双侧足三里穴。根据排出粪粒的性状，光照及黑暗时段的排便量及首粒黑便排出时间评价肠道传输功能及肠道运动的昼夜节律。光镜下观察脊髓及结肠组织的病理改变。通过透射电镜观察结肠cajal间质细胞（ICCs）超微结构；运用免疫组化法观察结肠per2、神经元型一氧化氮合成酶（nNOS）和ICCs的免疫染色分布表达；采用rt-PCR检测一个光照/黑暗周期24h的6个不同时点结肠per2基因表达水平以评估其生物钟节律。采用rt-PCR和Western Blot技术，分别检测结肠c-kit和nNOS基因和蛋白水平的表达。.结果表明：电针治疗能明显缩短脊髓损伤大鼠的首粒黑便排出时间，增加排便量、改善排出粪粒的性状及结肠组织形态结构。模型组肠道运动具有与假手术组同样的昼夜节律。光照下定时电针使光照时段首粒黑便的排出时间及排便量，与黑暗时段相比，没有显著性差异。模型组结肠per2基因表达的峰值出现在20:00，谷值出现在8:00；时间节律性与假手术大鼠基本一致。定时电针使结肠per2基因表达的最高峰值出现在白天12:00，并且12:00-16:00时段的基因表达高于其他时段，有别于假手术和模型组结肠per2基因的节律表达。但在整个光照黑暗周期内，电针大鼠结肠per2基因表达还保留了部分原有的生物节律。结肠Per2、nNOS和c-kit蛋白都主要表达在结肠肌间神经丛。电针能改善ICCs的超微结构，上调c-kit mRNA和蛋白的相对表达，下调nNOS mRNA和蛋白的相对表达。.结论：电针不会完全改变脊髓损伤大鼠结肠per2基因表达的生物钟节律，但会促使基因在电针后的一定时段内呈高表达。电针调节肠道运动的作用也有明显的时间性，与其促进结肠per2的基因表达时效性相关；可能与其通过per2对nNOS和c-kit的良性调控有关。电针能在避免过度进食对胃肠道造成负担的前提下，有效促进肠道运动；并且可以通过改变干预时间点来自主调节肠道运动的节律性。这一研究发现有重要的临床意义。