头针对脑缺血再灌注大鼠脑组织血管新生影响的相关机制研究

发布时间：2018-03-01 23:04

  本文关键词： 头针 血管新生 血管生成素-1(Ang-1) 血管内皮生长因子(VEGF) 环氧化酶-2(COX-2)　出处：《湖北中医药大学》2015年博士论文　论文类型：学位论文

【摘要】：脑卒中是目前严重危害我国中老年人生命与健康的疾病,本病突出表现为高发病率、高致残率、高死亡率、高复发率,每年我国有200万人新发脑卒中,150万人死于脑卒中,其中脑梗死占了全部脑卒中的60~80%,因其居高不下的致死率及致残率给国家和众多家庭造成沉重的经济和生活负担,故其已成为现代医学研究的热点课题。大量研究证明,缺血性脑损伤是一系列复杂的多环节的病理生理变化过程,早期主要以自由基产生、脑水肿、兴奋性氨基酸毒性为主;中期以炎症、神经元凋亡为主;后期以神经再生、血管新生和胶质细胞增殖等脑重构表现为主。脑缺血后,血管新生是脑微循环修复重建的一个重要病理生理过程。血管新生的程度与卒中后神经功能恢复的程度成正相关。血管内皮生长因子(VEGF)是目前发现的最强有力的血管形成因子。研究表明,脑缺血、缺氧可诱导血管内皮生长因子及其受体的表达,应用外源性VEGF可促进脑缺血半暗带新生血管的形成,并减少脑梗死体积;环氧化酶-2(COX-2)是花生四烯酸转化为前列腺素过程中的重要限速酶。近年来大量研究表明,COX-2是参与血管新生的重要因子之一;血管生成素(angiopoietins,Ang)是一族特异性作用于血管内皮细胞的生长因子,它们与血管内皮凋亡、血管形成后期的成熟、稳定性及重建方面均具有极大的相关性。近年来诸多研究均证实,三者对血管新生有重要作用,是近年来国际上兴起的新的研究领域,但其中有关中医药、针灸的促血管生成作用的研究相对较少,尤其是关于头针的促血管新生的研究鲜有报道。目的:根据上述分析,本研究在大鼠局灶性脑缺血模型的基础上,采用改良线栓法制备MCAO模型,拟“醒脑开窍,祛瘀生新”为治则,取顶颞后斜线、顶颞前斜线,通过观察头针对局灶性脑缺血再灌注大鼠缺血脑组织微血管超微结果的影响,以及对VEGF、COX-2、Ang-1三者表达的影响,从而探讨头针疗法对局灶性脑缺血再灌注损伤大鼠脑内血管新生影响的可能作用机制。方法:将清洁级sd雄性大鼠100只随机分为模型组(按缺血再灌注6h、24h、48h、96h分4组)、头针组(缺血再灌注6h、24h、48h、96h分别加电针刺激,分4组)、假手术组(大鼠麻醉切开颈部皮肤后,仅分离cca及ipa至ppa,不插线栓)、正常对照组(大鼠不予处理,正常给水给食),每组10只。各组大鼠采用zea-longa评分方法,随相应时相评定动物神经功能缺损。分别采用免疫组化sabc法检测脑缺血再灌注后各时间点因子Ⅷ相关抗原(fⅧr:ag)的表达,采用rt-pcr技术检测各组大鼠脑组织缺血半影区内ang-1mrna的表达水平,采用免疫组化法检测各组大鼠脑组织缺血半影区内vegf、es的表达水平,采用酶联免疫吸附(elisa)法、荧光定量pcr检测检测各组大鼠脑组织缺血半影区内cox-2的表达水平。结果:(1)大鼠局灶性脑缺血再灌注后,头针组和对照组大鼠均出现神经功能障碍,主要表现有:脑梗死灶对侧前肢内收、肩内旋,提尾悬空时左前肢不能伸直或紧贴胸壁,行走时向右侧转圈、倾倒,肌张力降低等。采用zea-longa评分方法,24h后头针组和对照组大鼠ci/rp后神经功能均逐渐恢复,但头针组较对照组大鼠恢复得更好,在96h时差异最为显著。(2)采用免疫组化sabc法检测fⅧr:ag,头针组与模型组比较,fⅧr:ag在脑微血管的表达在缺血后24h开始增加(p0.05)并持续上升,脑缺血后96h的差异具有极显著的统计学意义(p0.01),头针治疗随时间的延长可使其表达持续升高。由此可见头针治疗能够有效促进脑组织缺血半影区内微血管的新生,并且具有明显的累加蓄积效应。(3)采用免疫组化法检测ang-1mrna,正常对照组、假手术组ang-1mrna中等量表达,与模型组比较,头皮针组各时间点大鼠脑组织ang-1mrna含量均相对增多,其中脑缺血再灌注48hang-1mrna含量达高峰,与模型组比较,差异有统计学意义(p0.01,p0.05);(4)采用免疫组化法检测vegf及es,正常对照组、假手术组vegf中等量表达,与模型组比较,头皮针组各时间点大鼠脑组织vegf含量增多,es含量减少,其中脑缺血再灌注48hvegf含量达高峰,24hes含量达最低峰,与模型组比较,差异有统计学意义(p0.01,p0.05);(5)采用酶联免疫吸附(elisa)法、荧光定量pcr检测COX-2,正常对照组、假手术组COX-2少量表达,于模型组比较,头皮针组各时间点大鼠脑组织COX-2 mRNA及蛋白含量均减少,差异均有统计学意义(P0.05),其中脑缺血再灌注24h COX-2 mRNA含量、96h COX-2蛋白含量与模型组比较,差异最为显著(P0.01)。结论:取顶颞后斜线、顶颞前斜线,使用头针治疗局灶性脑缺血再灌注大鼠,可增加FⅧR:Ag、VEGF、Ang-1的表达,减少ES、COX-2的表达,从而促进脑组织缺血区血管新生,减少脑缺血区损伤,对缺血脑组织有保护作用。
[Abstract]:Stroke is a serious hazard for the elderly in our life and health disease, this disease especially the high incidence, high morbidity, high mortality, high recurrence rate, China has 2 million people a year in new stroke, 1 million 500 thousand people died of stroke, including cerebral infarction accounted for stroke 60~80%, because of its the high mortality rate and disability rate for the country and many families caused heavy economic burden and life, so it has become a hot topic of modern medical research. Many studies have shown that ischemic brain damage is the pathological change process of a series of complex multi link, mainly in the early production of free radicals, brain edema, mainly excited amino acid toxicity; in the middle of inflammation, neuronal apoptosis; late in nerve regeneration, angiogenesis and proliferation of glial cells in brain after cerebral ischemia. The manifestation of remodeling, angiogenesis is cerebral microcirculation reconstruction An important pathophysiological process. The recovery of nerve function and the degree of angiogenesis after stroke was positively correlated. Vascular endothelial growth factor (VEGF) has been found to be the most powerful angiogenic factor. The results show that cerebral ischemia, hypoxia induced vascular endothelial growth factor and its receptor expression, the formation of exogenous VEGF can promote the angiogenesis of ischemic penumbra, and reduce the volume of cerebral infarction; Cyclooxygenase -2 (COX-2) is four arachidonic acid into an important rate limiting enzyme in prostaglandin. Recent studies showed that COX-2 is one of the important factors involved in angiogenesis; angiopoietin (angiopoietins, Ang) is a growth factor a family of specific effects on vascular endothelial cells, and vascular endothelial apoptosis, angiogenesis of late maturity, stability and reconstruction are related greatly. In recent years a lot of research Study confirmed that the three plays an important role in angiogenesis, is a new research field in recent years, but the traditional Chinese medicine, study the angiogenic effect of acupuncture is relatively small, especially the study of few angiogenesis reports on scalp acupuncture. Objective: according to the above analysis, in this study based on the rat model of focal cerebral ischemia, the model of MCAO by using a modified suture method, proposed the "XNKQ, removing stasis and promoting new" rule, the D, D, to observe the effect of scalp acupuncture on focal cerebral ischemia reperfusion cerebral ischemia rat microvascular injection ultrastructural results, as well as VEGF, COX-2, Ang-1 expression of the three, and to explore the possible mechanism of acupuncture in the rat brain angiogenesis effect on focal cerebral ischemia reperfusion injury. Methods: male SD rats were randomly divided into model 100 Type group (by ischemia reperfusion 6h, 24h, 48h, 96h were divided into 4 groups (scalp acupuncture group), ischemia reperfusion 6h, 24h, 48h, 96h respectively with electroacupuncture stimulation, divided into 4 groups (sham operation group), rats were anesthetized open neck skin after separation of CCA and IPA to PPA only, do not plug suture), normal control group (rats without treatment, normal water feeding), 10 rats in each group. The rats by zea-longa score method, with the corresponding phase evaluation of animal neurologic defects. Were detected by immunohistochemical SABC method after cerebral ischemia reperfusion at different time points of factor VIII related antigen (f VIII r:ag) expression, the expression level of RT-PCR was used to detect the brain tissue of rats in ischemic penumbra of ang-1mrna, using immunohistochemical method to detect the rat brain tissue in ischemic penumbra of VEGF, the expression level of ES by enzyme-linked immunosorbent assay (ELISA) method, fluorescence quantitative PCR detection of cerebral tissue the rats in ischemic penumbra of Cox The expression of -2. Results: (1) in rats with focal cerebral ischemia reperfusion, scalp acupuncture group and control group rats showed neurological dysfunction, the main manifestations are: the focal cerebral infarct and contralateral forelimb shoulder adduction, internal rotation, the left forelimb hanging tail can not be straightened or close to the chest wall, when walking to right turn, dumping, muscle tension decreased. By zea-longa score method, 24h head acupuncture group and control group ci/rp rats after nerve function was restored gradually, but the scalp acupuncture group than in the control group rats recovered better in the 96h group was most significant. (2) using immunohistochemistry SABC method to detect f VIII r:ag, scalp acupuncture group compared with model group, the expression of r:ag in F VIII brain microvessels after ischemia 24h (P0.05) began to increase and continued to rise, with significant statistical difference in 96h after cerebral ischemia (P0.01), scalp acupuncture therapy with the extension of time can make its increased expression. Thus the head acupuncture treatment Therapy can effectively promote brain ischemic penumbra in angiogenesis, and has obvious effect. The cumulative volume (3) were detected by immunohistochemistry ang-1mrna, normal control group, sham operation group ang-1mrna expression amount, compared with the model group, scalp acupuncture group at different time points in rat brain tissue ang-1mrna content the relative increase in the content of cerebral ischemia reperfusion and reached the peak at 48hang-1mrna, compared with the model group, the difference was statistically significant (P0.01, P0.05); (4) immunohistochemical method was used to detect VEGF and ES, the normal control group, sham operation group VEGF expression amount, compared with the model group, scalp acupuncture group increased the content of VEGF in brain tissue of rats, ES content decreased, the content of cerebral ischemia reperfusion and reached the peak at 48hvegf, 24hes content reached the lowest peak, compared with the model group, the difference was statistically significant (P0.01, P0.05); (5) by enzyme-linked immunosorbent assay (ELISA) method, fluorescence Quantitative detection of PCR COX-2, normal control group, sham operation group, COX-2 expression in model group, scalp acupuncture group of COX-2 mRNA and protein in the brain tissue of rats were decreased, the differences were statistically significant (P0.05), 24h COX-2 in cerebral ischemia reperfusion mRNA content, 96h content of COX-2 protein compared with model the most significant difference between groups (P0.01). Conclusion: D, D, the use of scalp acupuncture on focal cerebral ischemia reperfusion in rats, increased F VIII R:Ag, VEGF, Ang-1 expression, decreased ES, COX-2 expression, so as to promote angiogenesis in ischemic brain tissue. To reduce the injury of cerebral ischemia area, has a protective effect on ischemic brain tissue.

【学位授予单位】：湖北中医药大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R245
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本文编号：1553952