针刺改善局灶性脑缺血脑微血管内皮细胞功能的动态观察

【作者】 赵万标；

【导师】 图娅；

【作者基本信息】 北京中医药大学 ， 针灸推拿学， 2004， 博士

【摘要】 研究目的:脑血管疾病以其高发病率、高死亡率、高致残率严重危害着人类的健康与生命。缺血性中风在脑血管病临床分类中占一半以上,并呈逐步增加趋势。正常状态下的脑毛细血管及包裹在其周围的星形胶质细胞构成了通透性极低的血脑屏障,只允许一些小分子物质通过;当缺血性中风发生时,微血管通透性发生显著改变,这种病理变化与脑内皮细胞损伤导致的功能变化有直接的关联。针刺能够显著改善缺血性中风模型的多项指标并参与脑血管内皮细胞损伤状态的调整和恢复,针刺十二井穴对局灶性脑缺血模型的治疗作用与干预时间和疗程选择有着极大的相关性。为了继续探寻这种现象的深层机理,本研究“针刺改善局灶性脑缺血脑微血管内皮细胞功能的动态观察”,采用大脑中动脉线栓阻塞法,通过大鼠局灶性脑缺血模型,检测缺血后不同时间点血管完整性标记成分的改变,以及这一过程中某些重要的粘附分子和细胞因子的表达变化,试图在分子水平探讨并阐明脑缺血再灌注脑损伤的炎性机理和针刺对局灶性脑缺血模型脑微血管内皮细胞功能的动态影响,旨在进一步探明针刺治疗缺血性中风过程中对血脑屏障通透性影响以及对脑内皮细胞保护作用,为临床治疗提供确切可行的干预时间及疗程选择等重要依据。研究方法:将健康老龄雄性 Wistar 大鼠采用大脑中动脉线栓阻塞法建立大鼠局灶性脑缺血模型;用免疫组化 SABC 法检测模型大鼠在脑缺血后治疗 ld、3d、5d、10d 时与内皮细胞功能有关的内皮素(ET-1)、FⅧ因子相关抗原(vWF)和细胞间粘附分子(ICAM-1)的表达,以标记微血管在脑缺血再灌注过程中损伤与修复变化;动态观察脑缺血后微血管形态结构和 ET-I、ICAM-1 等相关因子在微血管内皮细胞的表达变化规律,动态观察针刺井穴在不同时间点对上述指标的影响作用及其内在动态关联性。研究结果:1 实验大鼠神经症状行为改变 各组大鼠在造模后 1h 时神经症状行为均有阳性反应,与假手术组和空白对照组比较均有显著性差异(p<0.05),证明局灶性脑缺血实验模型成功;针刺不同疗程治疗各组大鼠在治疗前后的神经症状行为评定分析也有显著性差异(p<0.05),说明针刺十二井穴在本实验所设计的不同疗程内对PMCAO 大鼠神经症状的改善均具有显著作用。2 模型大鼠梗死局部脑组织的形态学改变 解剖造模大鼠,观察到缺血性中风灶位于颞叶及顶叶皮质区,与 MCA 支配的脑供血区域一致;十二井穴针刺不同时间组大鼠脑梗死范围均较模型组明显缩小。造模大鼠出现脑水肿等系列损伤表现,而针刺治疗各组脑缺血区周围组织损伤均有不同程度的减轻,其中治疗10天组最为显著。<WP=4>中文摘要 33 针刺对实验大鼠脑微血管内皮ⅧR:Ag 的影响针刺十二井穴后各时段组动物缺血半暗带表达ⅧR:Ag 的阳性细胞数均较同时段模型组增加,图像分析结果也显示缺血后各时段针刺组动物ⅧR:Ag 表达的总面积及积分光度均较同时段模型组显著增加,表明 针刺“十二井”穴能够有效促进缺血半暗带血管内皮细胞增生。FⅧ相关抗原主要在微血管内皮细胞中表达,在缺血后 3d开始增加(P<0.05),并持续上升;脑缺血针刺治疗组各个时间点的差异具有显著的统计学意义(P<0.01)4 针刺对实验大鼠 ET-1 的影响空白对照组、假手术组未见 ET-1 表达;模型组脑缺血后 1d 即出现表达, 5d达到表达高峰,各个时间点相比差异具有显著的统计学意义(P<0.01)。针刺后各时段组动物缺血半暗带表达 ET-1 的阳性细胞数均较同时段模型组减少,图像分析结果也显示缺血后各时段针刺组动物 ET-l 表达的总面积及积分光度均较同时段模型组显著减少,说明 “十二井穴”治疗在不同时间段能够有效抑制缺血半暗带血管内皮细胞 ET-l 的过度表达。5 针刺对实验大鼠 ICAM-1 的影响空白对照组、假手术组未见 ICAM-1 表达;模型组脑缺血后 1d 即出现表达,5d达到表达高峰,各个时间点相比差异具有显著的统计学意义 P<0.01。针刺十二井穴可有效下调脑缺血区 ICAM-1 表达,阻止缺血半暗带神经元的进一步损害,保护脑细胞。结论:通过本研究对针刺井穴在4个不同时段相关重要疗效指标的动态考察,可以认为:“十二井穴”针法可使缺血半暗区相关神经因子的表达发生良性变化;激活内皮细胞,促进脑血管内皮细胞损伤的修复;提高脑血管自动调节功能;保护正常脑组织、防止缺血中心区坏死区扩大具有显著的临床意义。同时,本研究结果提示,针刺井穴对脑缺血状态下微血管内皮细胞功能的全方位和多层面良性调节机制,并且这种良性机制随着干预时间的选择(早期/6d)和疗程的适度延长(5天以上),具有明显的累加蓄积效应。相信这一研究结果将会对临床的缺血性中风治疗研究提供具有指导性价值的参考。更多还原

【Abstract】 Research purpose: Cerebrovascular disease with high disability rate, lethality and deathrate endanger human health and life seriously. Cerebral ischemia apoplexy accounts forover half clinical classifying at the cerebrovascular disease,show a tendency to increaseprogressively. The brain capillary and star glue quality cell which enwrap around it under thenormal condition form the permeation extremely low blood-brain barrier(BBB), onlypermit some little molecule to pass; When cerebral ischemia apoplexy happen, theremarkable changes takes place in the permeation of the blood capillary, this kind ofpathology change have direct relation to the change of the function of the brain endothelialcell damaged. Acupuncture can improve the multiple indexes of cerebral ischemia apoplexy modeland participate in the adjustment and recovery of the cerebrovascular endothelial celldamaged, acupuncture 12 well point have great relation to the treatment function andintervene time and course of treatment choose in the local cerebral ischemia model.Continuing to seek the deep mechanism of phenomenon, this research " The dynamicobservation of the acupuncture improve the function of capillary vessel’s endothelial cellof local cerebral ischemia model ", adopts middle cerebral artery occlusion method,through the local cerebral ischemia model of rat, detects the change of the blood vesselintegrality marked composition of different time after the lacking blood, and the change ofthe some important expression which adhension molecules and cell factors, attempt todiscuss the inflammation mechanism of the reperfusion of cerebral ischemia and thedynamic effect of the acupuncture improve the function of capillary vessel’s endothelialcell of local cerebral ischemia model in the probe level, purposes to prove up the effect ofacupuncture treat the permeation of the BBB and acupuncture protect the endothelial cellin the progress of the cerebral ischemia apoplexy, provides the important feasibility gist ofthe intervene time and the treatment choose in the clinical treatment.Research method: The health old male rats are adopted the MCAO method to create therat’s local cerebral ischemia-reperfusion model, examines the expression of the ET-1,Vwfand ICAM-1in the model of rat after cerebral ischemia treatment in 1d,3d,5d,10d with theimmunohistochemical SABC method, marks capillary vessel the vary of the injury andrepair in the progress of the cerebral ischemia-reperfusion; at the same time examines theexpression of IL-1β、IL-6 at each time point with immunohistochemical S-P method,observes the appearance construction of capillary vessel after cerebral ischemia and thevariety regulation of these inflammatory factories expression in capillary vesselendothelial cell.Reseach result:1, The behavioral change of the experimental rat’s neural symptom The neural symptom behaviors of every group rat have positive reaction at an hourafter the model created, the sham-operated control group and the control group havesignificance difference (p<0.05), prove the success of the local cerebral ischemia model;<WP=6>英文摘要 5the analyse of every group rat’s neural symptom behavior of in the different course ofacupuncture treatment have significance difference,too(p<0.05), prove acupuncture 12well point have remarkable function to the PMCAO rat’s neural symptom improvement inthe different course of treatment designed in this experiment.2, the morphologic change of the infarct of the model rat’s local brain organizesDissecting model rat, we observe cerebral ischemia apoplexy that lies in the temporal lobeand terminal leaf cortex district, keep the same with brain blood supply area that MCAcontrols; the brain infarct range of 12 well point acupuncture different time group ratobviously reduce than the model group. model rat appear brain hydroncus ,etc. the injuryis mitigated by acupunctur更多还原