【作者】 崔瑾；

【导师】 严洁；

【作者基本信息】 湖南中医药大学 ， 针灸推拿学， 2007， 博士

【副题名】内关穴位埋针对心肌缺血小型猪血管新生相关生长因子影响的实验研究

【摘要】 大量临床和实验研究已经发现,针刺手厥阴心包经内关穴有改善冠心病患者心功能,改善心肌能量代谢障碍、减少氧自由基生成、抑制钙超载、调整血管内源性保护物质等作用,但未见从血管新生角度研究不同经脉(穴)刺激治疗心肌缺血损伤的调节机制。本实验试图从与心相关的心包经及与心无直接联系但与“血”有关的膀胱经背俞穴对心肌缺血损伤调整作用予以对比研究。目的:在内关埋针确能促进血管密度增加的基础上,采用与血管新生相关的功能性基因芯片技术,观察缺血性损伤心肌血管生长因子相关分子功能类别的mRNA水平的表达整体特征,并选择其中重要因子从基因、蛋白水平研究“内关”穴位埋针促心肌缺血损伤小型猪血管新生调节机制,并以膀胱经(穴)“膈俞”作对比研究,观察两者血管新生差异性,以进一步探讨手厥阴心包经(内关)与心相关、阐述经脉脏腑相关相对特异性。方法:32只小型猪随机分为4组,分别为假手术组、模型组、内关组和膈俞组,采用左冠状动脉前降支结扎建立心肌缺血模型,假手术组穿线不结扎,内关组和膈俞组分别在模型组基础上进行内关、膈俞穴埋针治疗,以内关穴位埋针作为主要观察对象,与膈俞穴位埋针进行比较,观察心电图ST段、心肌组织形态和超微结构变化,免疫组化方法测定心肌毛细血管密度,血管生成功能性基因芯片找出差异基因,用实时定量PCR和Western blot方法验证和分析血管内皮细胞生长因子(VEGF)、转化生长因子β3(TGF-β3)的基因和蛋白表达。结果:①心肌缺血损伤时心电图ST段降低,内关和膈俞穴位埋针有不同程度的促进心电图ST段的恢复,与模型组比差异均有显著性(P＜0.05),但内关组优于膈俞组(P＜0.05)。②内关穴埋针能更好地促进缺血损伤心肌组织形态和超微结构的修复,增加心肌毛细血管密度,与模型组比差异非常显著(P＜0.01),膈俞也有一定的改善作用(P＜0.05),但内关组优于膈俞组(P＜0.05)。③Q Series血管生成功能性芯片结果示:在心肌血管新生差异表达大于2倍的基因数目上,显示内关组的变化比膈俞组大,而其功能分类中特异的促进因子和抑制因子、血管内皮生长因子、成纤维细胞生长因子及其受体、细胞因子和化学增活素、转录因子等是各组间共性,其中血管内皮细胞生长因子及受体(VEGF、VEGFR)、转化生长因子β(TGFβ)、血管生成素-1(Angiopoietin 1),成纤维细胞生长因子及受体(FGF、FGFR)、血小板衍生生长因子(PDGF)等又是其共同表达,且表达在2倍以上。④内关穴埋针能上调VEGF基因和蛋白表达,与模型组比较差异非常显著(P＜0.01),膈俞也有促进VEGF基因表达上调的趋势,但与模型组比差异无显著性(P＞0.05),膈俞与内关同样具有促进VEGF蛋白表达上调的作用(P＞0.05)。⑤内关穴埋针能上调TGF-β3基因和蛋白表达,与模型组比较差异非常显著(P＜0.01),膈俞有促进TGF-β3基因表达上调的趋势,但与模型组比差异无显著性(P＞0.05),内关上调TGF-β3蛋白表达的作用强于膈俞(P＜0.05)。结论:①内关穴位埋针可能通过上调VEGF基因和蛋白表达,上调TGF-β3基因和蛋白表达,破坏血管生成的平衡系统,增强血管形成能力,增加毛细血管密度,促进侧枝循环建立,增加缺血心肌血流,实现对心肌组织的保护作用,表现为促进心电活动、心肌组织形态和超微结构的恢复。②内关穴埋针对心肌细胞有较明显的保护作用,可能是经脉(穴)—脏腑相关相对特异性的依据之一。更多还原

【Abstract】 It has been demonstrated by many clinical studies that acupuncture Neiguan can improve heart function.The previous animal experimental research has indicated that electro-acupuncture can ameliorate energy metabolism dysfunction of rat ischemia-reperfusion injured myocardium,decrease the production of reactive oxygen species,inhibit calcium overload and increase endogenous protective substances.But the regulation mechanism that different meridians point treat injury of myocardial reperfusion across blood vessels neogenesis has not been found.This experiment tries to give contrast a research that pericardium meridian and urinary bladder meridian adjust to myocardial ischemia injure.[Objective]At basis that neiguan needle implant can surely promote the afferent density to increase,observe the whole mRNA express characteristic of related member function of vascular growth factor in ischemic injury myocard. by functioning gene array technique.Moreover select important factor and investigate the mechanism of regulation of blood vessels neogenesis in ischemia injured pig myocardium by needle implant Neiguan in gene and protein level,Points Geshu were chosen as control in order to observe blood vessels neogenesis variability,explore the relative specificity of correlations between Pericardium Channel of Hand-Jueyin(point Neiguan) and the Heart, Meridians and viscera.[Methods]32 Guizhou miniswine were randomly divided into four groups including pseudo-operation group、model group、needle implant on Neiguan group、needle implant on Geshu group.Every group have eight.The animal model of myocardium ischemia was set up by ligation of coronary artery.After needle implant Neiguan,Geshu respectively,changes of ST segmen were observed,changes of myocardium morphology observed by optical and electron microscope,detect heart muscle capillary by immunohistochemistry method,discrepancy genes were searched by functioning gene array technique of correlations blood vessels neogenesis,check and analyze mRNA and protein expressions of VEGF and TGF-β3 by Real time PCR and Western blot methods.[Results]①ST segment degraded after ischemia injury.needle implant Neiguan and Geshu can both promote the recoveried ST segment,showed obvious difference from modle group(P＜0.05),but Neiguan group surpassed Geshu group(P＜0.05).②Needle implant Neiguan can accelerate the repairment of histomorphology and ultrastructure of ischemia injured myocardium,increase myocardium capillary density,showed obvious difference from modle group(P＜0.01). Needle implant Geshu had amelioration effects,but Neiguan group surpassed Geshu group(P＜0.05)③the results of Q Series functioning gene array of correlations blood vessels neogenesis suggested:in the way of genes numbers that discrepancy express exceeded two times in myocardium blood vessels neogenesis,changes in Neiguan group exceed to Geshu group,Specific Promoters and Inhibitors, Vascular Endothelial Growth Factors,Fibroblast Growth Factors & Receptors, Cytokines and Chemokines and transcription Factors are common in each group,VEGF、VEGFR、TGFβ、Angiopoietin 1、FGF、FGFR and PDGF are co-expression,furthermore express are above two times.④Needle implant Neiguan can upregulate the expression of VEGF mRNA and protein,showed obvious difference from modle group(P＜0.01).Needle implant Geshu showed a tendency to upregulate the expression of VEGF mRNA,but had not obvious difference from the modle group(p＞0.05),Geshu can also upregulate the expression of VEGF protein,which were not obviously different from the Neiguan group(p＞0.05).⑤Needle implant Neiguan can upregulate the expression of TGF-β3 mRNA and protein,showed obviously difference from modle group(P＜0.01).Needle implant Geshu showed a tendency to upregulate the expression of TGF-β3 mRNA,but had not obvious difference from the modle group(p＞0.05),effect that Neiguan upregulate the expression of TGF-β3 protein,is better to Geshu group(p＜0.05)[Conclusions]①Needle implant Neiguan may protect myocardium tissue through the way of upregulating the mRNA and protein expression of VEGF and TGF-β3 mRNA, destroying balance system of Angiogenesis,enhancing the ability of angiopoiesis,increasing capillary density,promoting establishment of collateral circulation,increasing blood flow of ischemic myocardial.Such protective effect was manifested as improving the electrophysiology and accelerating the repairment of histomorphology and ultrastructure of pig ischemian injured myocardium.②The strong myocardium-protective effect of Needle implant Neiguan probably is one evidence of the relative specificity of the Meridians and viscera..更多还原