本文關(guān)鍵詞： 頭針 血管新生 血管生成素-1(Ang-1) 血管內(nèi)皮生長因子(VEGF) 環(huán)氧化酶-2(COX-2)　出處：《湖北中醫(yī)藥大學(xué)》2015年博士論文　論文類型：學(xué)位論文

【摘要】：腦卒中是目前嚴(yán)重危害我國中老年人生命與健康的疾病,本病突出表現(xiàn)為高發(fā)病率、高致殘率、高死亡率、高復(fù)發(fā)率,每年我國有200萬人新發(fā)腦卒中,150萬人死于腦卒中,其中腦梗死占了全部腦卒中的60~80%,因其居高不下的致死率及致殘率給國家和眾多家庭造成沉重的經(jīng)濟(jì)和生活負(fù)擔(dān),故其已成為現(xiàn)代醫(yī)學(xué)研究的熱點(diǎn)課題。大量研究證明,缺血性腦損傷是一系列復(fù)雜的多環(huán)節(jié)的病理生理變化過程,早期主要以自由基產(chǎn)生、腦水腫、興奮性氨基酸毒性為主;中期以炎癥、神經(jīng)元凋亡為主;后期以神經(jīng)再生、血管新生和膠質(zhì)細(xì)胞增殖等腦重構(gòu)表現(xiàn)為主。腦缺血后,血管新生是腦微循環(huán)修復(fù)重建的一個(gè)重要病理生理過程。血管新生的程度與卒中后神經(jīng)功能恢復(fù)的程度成正相關(guān)。血管內(nèi)皮生長因子(VEGF)是目前發(fā)現(xiàn)的最強(qiáng)有力的血管形成因子。研究表明,腦缺血、缺氧可誘導(dǎo)血管內(nèi)皮生長因子及其受體的表達(dá),應(yīng)用外源性VEGF可促進(jìn)腦缺血半暗帶新生血管的形成,并減少腦梗死體積;環(huán)氧化酶-2(COX-2)是花生四烯酸轉(zhuǎn)化為前列腺素過程中的重要限速酶。近年來大量研究表明,COX-2是參與血管新生的重要因子之一;血管生成素(angiopoietins,Ang)是一族特異性作用于血管內(nèi)皮細(xì)胞的生長因子,它們與血管內(nèi)皮凋亡、血管形成后期的成熟、穩(wěn)定性及重建方面均具有極大的相關(guān)性。近年來諸多研究均證實(shí),三者對血管新生有重要作用,是近年來國際上興起的新的研究領(lǐng)域,但其中有關(guān)中醫(yī)藥、針灸的促血管生成作用的研究相對較少,尤其是關(guān)于頭針的促血管新生的研究鮮有報(bào)道。目的:根據(jù)上述分析,本研究在大鼠局灶性腦缺血模型的基礎(chǔ)上,采用改良線栓法制備MCAO模型,擬“醒腦開竅,祛瘀生新”為治則,取頂顳后斜線、頂顳前斜線,通過觀察頭針對局灶性腦缺血再灌注大鼠缺血腦組織微血管超微結(jié)果的影響,以及對VEGF、COX-2、Ang-1三者表達(dá)的影響,從而探討頭針療法對局灶性腦缺血再灌注損傷大鼠腦內(nèi)血管新生影響的可能作用機(jī)制。方法:將清潔級sd雄性大鼠100只隨機(jī)分為模型組(按缺血再灌注6h、24h、48h、96h分4組)、頭針組(缺血再灌注6h、24h、48h、96h分別加電針刺激,分4組)、假手術(shù)組(大鼠麻醉切開頸部皮膚后,僅分離cca及ipa至ppa,不插線栓)、正常對照組(大鼠不予處理,正常給水給食),每組10只。各組大鼠采用zea-longa評分方法,隨相應(yīng)時(shí)相評定動(dòng)物神經(jīng)功能缺損。分別采用免疫組化sabc法檢測腦缺血再灌注后各時(shí)間點(diǎn)因子Ⅷ相關(guān)抗原(fⅧr:ag)的表達(dá),采用rt-pcr技術(shù)檢測各組大鼠腦組織缺血半影區(qū)內(nèi)ang-1mrna的表達(dá)水平,采用免疫組化法檢測各組大鼠腦組織缺血半影區(qū)內(nèi)vegf、es的表達(dá)水平,采用酶聯(lián)免疫吸附(elisa)法、熒光定量pcr檢測檢測各組大鼠腦組織缺血半影區(qū)內(nèi)cox-2的表達(dá)水平。結(jié)果:(1)大鼠局灶性腦缺血再灌注后,頭針組和對照組大鼠均出現(xiàn)神經(jīng)功能障礙,主要表現(xiàn)有:腦梗死灶對側(cè)前肢內(nèi)收、肩內(nèi)旋,提尾懸空時(shí)左前肢不能伸直或緊貼胸壁,行走時(shí)向右側(cè)轉(zhuǎn)圈、傾倒,肌張力降低等。采用zea-longa評分方法,24h后頭針組和對照組大鼠ci/rp后神經(jīng)功能均逐漸恢復(fù),但頭針組較對照組大鼠恢復(fù)得更好,在96h時(shí)差異最為顯著。(2)采用免疫組化sabc法檢測fⅧr:ag,頭針組與模型組比較,fⅧr:ag在腦微血管的表達(dá)在缺血后24h開始增加(p0.05)并持續(xù)上升,腦缺血后96h的差異具有極顯著的統(tǒng)計(jì)學(xué)意義(p0.01),頭針治療隨時(shí)間的延長可使其表達(dá)持續(xù)升高。由此可見頭針治療能夠有效促進(jìn)腦組織缺血半影區(qū)內(nèi)微血管的新生,并且具有明顯的累加蓄積效應(yīng)。(3)采用免疫組化法檢測ang-1mrna,正常對照組、假手術(shù)組ang-1mrna中等量表達(dá),與模型組比較,頭皮針組各時(shí)間點(diǎn)大鼠腦組織ang-1mrna含量均相對增多,其中腦缺血再灌注48hang-1mrna含量達(dá)高峰,與模型組比較,差異有統(tǒng)計(jì)學(xué)意義(p0.01,p0.05);(4)采用免疫組化法檢測vegf及es,正常對照組、假手術(shù)組vegf中等量表達(dá),與模型組比較,頭皮針組各時(shí)間點(diǎn)大鼠腦組織vegf含量增多,es含量減少,其中腦缺血再灌注48hvegf含量達(dá)高峰,24hes含量達(dá)最低峰,與模型組比較,差異有統(tǒng)計(jì)學(xué)意義(p0.01,p0.05);(5)采用酶聯(lián)免疫吸附(elisa)法、熒光定量pcr檢測COX-2,正常對照組、假手術(shù)組COX-2少量表達(dá),于模型組比較,頭皮針組各時(shí)間點(diǎn)大鼠腦組織COX-2 mRNA及蛋白含量均減少,差異均有統(tǒng)計(jì)學(xué)意義(P0.05),其中腦缺血再灌注24h COX-2 mRNA含量、96h COX-2蛋白含量與模型組比較,差異最為顯著(P0.01)。結(jié)論:取頂顳后斜線、頂顳前斜線,使用頭針治療局灶性腦缺血再灌注大鼠,可增加FⅧR:Ag、VEGF、Ang-1的表達(dá),減少ES、COX-2的表達(dá),從而促進(jìn)腦組織缺血區(qū)血管新生,減少腦缺血區(qū)損傷,對缺血腦組織有保護(hù)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.

【學(xué)位授予單位】：湖北中醫(yī)藥大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R245
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本文編號：1553952