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电项针对大鼠脑缺血再灌注脑损伤模型的缺氧诱导因子（HIF-1α）及其靶基因的影响研究
Experimental Studies on Effects of Electroacupuncture on the Nuchal Region on the Expression of HIF-1α and It’s Relative Target Genes Following Cerebral Ischemia Reperfusion in Rats
【作者】 杨续艳；
【导师】 高维滨；
【作者基本信息】 黑龙江中医药大学 ， 针灸推拿学， 2009， 博士
【摘要】 缺血性脑血管病是临床的常见病、多发病，随着溶栓疗法在临床的大量应用，再灌注损伤的问题越来越受到重视。如何减轻脑缺血再灌注后组织损伤，提高神经细胞对缺血缺氧的耐受，已经逐渐成为研究热点。项针在脑缺血急性期和恢复期的治疗方面取得了显著疗效，但对于项针疗效机理的实验研究则刚刚开始。本课题从电项针干预大鼠脑缺血再灌注后缺血半暗带缺氧诱导因子(HIF-1α)及相关靶基因表达的研究入手，为针刺治疗缺血性脑血管病的机制研究提供新的研究方向。目的：观察大鼠脑缺血再灌注(CIR)后缺血半暗区缺氧诱导因子(HIF-1α)和相关靶基因表达的变化规律及电项针对其的影响，探讨电项针治疗缺血性脑血管病的作用机制。方法：采用改良线栓法制备大鼠大脑中动脉缺血再灌注模型。将Wistar雄性大鼠，随机分成4组：假手术组(A)、模型组(B)、电体针组(C)及电项针组(D)，各组随机分成1 2h、1d、3 d、5d四个时间段，对大鼠进行实验观察：(1)参照Zea-Longa的5级评分标准观察实验各组大鼠在再灌注后各时间点神经功能缺损评分；(2)HE染色观察各组大鼠脑组织形态学改变情况；(3)电镜观察各组大鼠脑组织超微结构的改变；(4)应用免疫组织化学、原位杂交技术检测大鼠局灶性脑缺血再灌注后缺血半暗带HIF-1α蛋白及mRNA、EPO蛋白及mRNA、GLUT-1蛋白及mRNA和VEGF蛋白的表达变化以及观察电体针、电项针治疗对这些基因表达的影响。结果：1．模型组脑缺血再灌注后出现明显神经功能缺损，与假手术组比较差异显著(P＜0．01)，证实MCAO实验模型成功；再灌注3d、5d时电体针组、电项针组与模型组比较差异显著(p＜0．05，p＜0．01)，电项针与电体针比较，电项针组评分少于电体针组，具有显著差异(p＜0．05)，说明电项针对大鼠神经功能缺损的恢复有明显的促进作用。2．HE染色显示：与模型组比较，电项针组和电体针组缺血半暗带神经细胞肿胀较轻，神经元数量增多，新生毛细血管增多，电项针组较电体针组改善明显。3．电镜观察显示：与模型组比较电项针组和电体针组神经细胞胞体内，可见丰富的高尔基复合体，线粒体和其它细胞器结构完整，突触膜活性区明显增多，突触小泡增加。其中，电项针组结构改变明显好于电体针组。4．免疫组织化学结果显示：与模型组比较，电项针和电体针治疗可提高HIF-1α、EPO、GLUT-1、VEGF蛋白表达水平(p＜0．01)，同时间点电项针组与电体针组比较有显著性差异(p＜0．01或p＜0．05)。5．原位杂交结果显示：与模型组比较，电项针和电体针治疗可提高HIF-1αmRNA、EPOmRNA、GLUT-1 mRNA的表达(p＜0．01)，而电项针组与同时间点电体针组比较有显著性差异(p＜0．01或p＜0．05)。结论：1．电项针较电体针治疗可更好地增加梗死灶周围脑组织供血，挽救脑缺血半暗带神经细胞，达到改善神经功能的作用。2．电项针较电体针治疗可明显促进缺血半暗带胶质细胞和毛细血管增生，减轻神经元损伤。3．电项针较电体针治疗缺血半暗神经细胞的超微结构改善明显，突触膜活性区明显增多，突触小泡增加。4．电项针治疗可能通过提高HIF-1α(mRNA)、EPO(mRN)、GLUT-1(mRNA)、VEGF蛋白表达，达到营养神经、血管重构、抑制兴奋氨基酸释放和恢复能量供应的作用，从而提高神经细胞对缺血缺氧的耐受，减少脑缺血再灌注损伤。
【Abstract】 As a familiar disease,Apoplexy has taken place frequently and the ratioof death and lameness are both very high.With the application ofthrombolytic therapy,the reperfusion injury is paid to more and moreattention.How to reduce cerebral ischemia-reperfusion injury and enhanceneuronal tolerance to ischemia and hypoxia,has gradually become a researchhotspot.The elctro-acupuncture on nuchal region(EANR)n the acute stageof cerebral ischemia and effects of the treatment period achieved significantefficacy,but efficacy of the mechanism of the needle is just beginning.Theissue of needle-intervention from the power of hypoxia-inducible factor(HIF-1α)and related gene expression in the target to start for theacupuncture treatment of ischemic encephalopathy study provides a newmechanism for the research.Objective:To observe the expression of HIF-1αand it’s correlative geneof in ischemic penumbra in rats after acute cerebral infarction and to explorethe effect and the possible mechanism of electro-nape acupuncture therapy.Methods:The model of focal cerebral ischemia was made by blockingthe middle cerebral artery with monofilament in rats.The middle cerebralartery ocelussion rats were randomly divided into 4 groups:sham group(group A),model group(group B),electro-acupuncture group(group C)and electro-nape acupuncture group(group D)Male Wistar rats weredivided randomly into four groups:sham group,model group,electro-napeacupuncture group and electro-acupuncturegroup;each group was separatedinto 12h,1d,3d,5d after rerperfusion as subgroup for investigation.HEstaining and electron Electron microscope were used to look into the changesof histomorphology.Took Immunohisto chemistry and In situ hybridizationto detect the expression of HIF-1α(mRNA)、EPO(mRNA)、GLUT-1(mRNA),and VEGF observe ultrastructure in brain tissue、nerve functionscore. Results:Electro-nape acupuncture has the obvious auxoaction onrecuperation of neurologic impairmentThe results showed electro-napeacupuncture possessed protective effect against cerebral ischemia-reperfusion injury,compared with the model group.The results indicatedthat compared with group B,the neuron ultrastructure was mended in groupC and in group D(P＜0.05,P＜0.01)It was more dominant in group D thanthat in group C(P＜0.05)Electro-nape acupuncture can lessen neurologicimpairment.Results pointed out,that compared with the model group,electro-acupuncture group and electro-nape acupuncture can increase theexpression of HIF-1α(mRNA)、EPO(mRNA)、GLUT-1(mRNA)andVEGF.Conclusion:1.Electro-nape acupuncture treatment to save the cerebral ischemicpenumbra of nerve cells around the infarction to enhance blood supply tobrain tissue,to improve nerve function.2.Electro-nape acupuncture can promote ischemic penumbra glial cellsand capillary hyperplasia,reduce neuronal damage and improve theultrastructure of nerve cells.3.Electro-nape acupuncture of ischemic penumbra of the ultrastructure ofnerve cells improved synaptic membrane activezone increased significantly,to increase synaptic vesicles.4.Electro-nape acupuncture treatment by increasing HIF-1α,EPO,GLUT-1,VEGF protein expression to the nutritional nerves,vascularremodeling and restoration of the role of energy supply,thereby enhancingthe nerve cells of hypoxic-ischemic tolerance,reducing cerebralischemia-reperfusion injury.5.Electro-nape acupuncture treatment by increasing HIF-1αmRNA,EPOmRNA,GLUT-1mRNA expression to the nutritional nerves,vascularremodeling and restoration of the role of energy supply,thereby enhancingthe nerve cells of hypoxic-ischemic tolerance,reducing cerebral ischemia-reperfusion injury.