【作者】 王恩龙；

【导师】 马智；

【作者基本信息】 辽宁中医药大学 ， 中医内科， 2008， 博士

【摘要】 头针对急性脑缺血再灌注模型大鼠神经元保护机制的研究缺血性脑血管病是目前严重危害人类健康的疾病,具有高发病率、高死亡率、高致残率的特点。中医药防治中风具有悠久的历史,早在2000多年前的中医经典著作《黄帝内经》中已经有关于中风的病因病机、治疗原则、预防保健等方面的记载,尤其在针灸治疗中风病方面,历代医学著作均有丰富论述。近年来临床研究表明针刺头穴顶颞前斜线、顶颞后斜线能够改善患侧肢体的运动能力,改善患者的神经功能缺损的评分。本项研究采用线栓法制备大鼠大脑中动脉缺血再灌注模型(MCAO),通过电镜、原位杂交、免疫组化、RT-PCR、ELISA等方法系统观察了针刺顶颞前斜线对大脑中动脉缺血再灌注模型大鼠脑损伤的保护作用,并且通过细胞凋亡、凋亡相关基因表达、炎症反应以及超微结构等几个方面探讨针刺抗脑缺血的作用机制,为临床头针治疗缺血性脑血管病提供了科学的实验依据。实验方法:选择健康清洁SD大鼠,随机分为A组(假手术组)、B组(模型组)、C组(头针组)。参照Longa的改良的线栓方法制作大鼠局灶性脑缺血再灌注(MCAO)模型。应用TTC染色,观察缺血再灌注后24小时梗死体积,缺血再灌注后3、24、72小时神经功能缺损评分;应用TUNEL法观察神经元凋亡;应用免疫组化法观察脑缺血再灌注后各时间点bcl-2、bax基因表达变化;应用ABC-ELISA法检测IL-1β;应用RT-PCR技术观察脑缺血再灌注后各时间点caspase-3mRNA、caspase-8mRNA表达;光镜、电镜观察神经元结构。所有实验数据用均数±标准差(x±s)表示,应用SPSS10.0统计软件进行数据分析。实验结果:1、脑梗死体积。A组未发现梗死灶,B组梗死灶在额、顶叶皮质及尾状核、壳核区域,C组梗死区域小于B组,有统计学意义(P<0.05)。2、神经功能缺损评分。C组在脑缺血再灌注后3、24、72小时神经功能缺损评分与B组比较均有降低,其中缺血再灌注后24、72小时,C组与B组比较有统计学意义(P<0.01、P<0.05)。3、神经元凋亡结果。A组可见少量凋亡细胞,B组、C组凋亡细胞主要分布在梗死周围区域,即半暗带区。凋亡细胞在再灌注后3小时开始增加,24小时达高峰,72小时凋亡细胞数减少;B组、C组缺血再灌注后各时间点凋亡细胞与A组比较有显著性差异(P<0.01);C组缺血再灌注后各时间点凋亡细胞与B组比较,显著减少(P<0.01、P<0.05)。4、免疫组化结果。A组可见少量bcl-2、bax基因表达,B组、C组bcl-2、bax基因表达在半暗带区。bcl-2、bax基因表达在缺血再灌注后3小时开始增强,24小时达高峰,bcl-2高峰表达持续至72小时,bax表达在再灌注后72小时稍有下降;C组再灌注后各时间点bcl-2表达增强,bax表达下降,与B组比较有统计学差异。5、ABC-ELISA法检测IL-1β。A组IL-1β在大脑皮质少量表达,B组在再灌注早期IL-1β大量出现,72小时达到高峰,C组在各时间点IL-1β显著下降,与B组比较有显著性差异。6、RT-PCR结果。A组caspase-3mRNA、caspase-8mRNA仅见微弱表达,B组caspase-3mRNA、caspase-8mRNA随着缺血再灌注时间延长,表达逐渐增强,再灌注后24小时表达达到高峰,以后逐渐减弱,C组各时间点caspase-3mRNA、caspase-8mRNA表达与B组比较显著降低。7、超微结构变化。A组神经元、神经胶质细胞以及毛细血管结构完整,B组神经元细胞质内可见大量空泡样变,细胞器消失;胶质细胞胞质内大量空泡样改变;毛细血管管腔变小,血管内皮细胞增大。C组半暗带区大脑皮质神经元细胞胞质内空泡变少,细胞器较丰富,可见粗面内质网、线粒体、核糖体等,核大,染色质较均匀;胶质细胞胞质内粗面内质网轻度扩张,高尔基体增多;毛细血管管腔变大,内皮光滑。结论:第一、针刺头穴能够改善MCAO大鼠神经功能缺损评分,对保持缺血区神经元结构和功能的完整具有重要作用。第二、针刺头穴能够减少MCAO大鼠缺血区神经细胞凋亡数量。第三、针刺头穴能够上调MCAO大鼠缺血区bcl-2表达,下调bax表达。第四、针刺头穴能够抑制MCAO大鼠缺血区炎症反应,下调白介素1β表达。第五、针刺头穴能够抑制凋亡启动因子caspase-3、caspase-8,从而抑制细胞凋亡。本项研究表明针刺头穴能够从神经细胞超微结构、细胞凋亡、炎症反应等方面证实头穴针刺能够对MCAO所致脑损伤有保护作用,为临床推广和普及头针疗法以及深入研究提供实验依据。更多还原

【Abstract】 Preface:Nowdays the ischemia cerebral vascular disease has threatened the health of human being. The ischemia vascular disease has the character of high morbidity, high mortality rate and high cripples rate. Chinese medicine has long history of preventing and controlling stroke. The Chinese medicine classic work《the yellow Emperor Canon of Internal Medicine》has the record of cause, treatment principle and Prevention health care for stroke. Especially acupuncture were used to treat stroke. Recently Clinical researches demonstrate that puncturing the skull point can improve moving ability of the paralytic limb and make the scale of the patient neurology dysfunction better. The MCAO rats were adopted in this research and the technology of TUNEL、RT-PCR、ELISA、immunohistochemistry and electron-microscope were used. the preventing effect of puncturing the head point were observed systematically and the mechanism of the acupuncture anti cerebral ischemia were probed through the cell apoptosis, the express of the associated apoptotic gene, inflammation reaction and neuronal micro structure. This research had provided the scientific evidence for the head needle treating the ischemia cerebral vascular disease clinically.Materials and Methods:The health rats were randomly divided into 3 groups. A group(sham-operated group);B group(ischemia/reperfusion group);C group (acupuncture treatment group). The middle cerebral artery occlulsion(MCAO) were established by using the longas method. The infarct volume were studied by TTC staining after 24 hours of ischemia and reperfusion, scores of neurological functions deficits were evaluated after 3h、24h、72h following ischemia /reperfusion, neuronal apoptosis were detected by the TUNEL staining, the expression of bcl-2 and bax were observed by immunohistochemistry after 3h、24h、72h following ischemia /reperfusion, the IL-1βwas detected by using the ELISA method, the expression of caspase-3mRNA、caspase-8mRNA were detected by RT-PCR methods, the neural micro structure was observed by electron microscope. All data were expressed as mean±SD. Statistical analysis were carried out by spss10.0.Results:1、Infarct volumesThere were not infarcts volume in A group. The infarcts in B、C group were mainly in frontal-parietal cortex, caudates nucleus and putamen nucleus. The infarcts volumes of C group were significantly less than B group(P<0.05)2、Evaluation of neurological functions deficits Scores of neurological functions deficits of C group were less than that of B group after 3h、24h、72h following ischemia/reperfusion. Compared with the Scores of neurological functions of B group, the difference between C group and B group is significant after 24h、72h following ischemia/reperfusion.3、Neuronal apoptosisApoptotic neurons were mainly located around ischemic tissues, that was ischemic penumbra. There were few apoptotic neuronal cells in A group. The numbers of apoptotic cell increased after 3h following cerebral ischemia/reperfusion, then gradually increased as time went on, at 24h reached the peak, then decreased gradually. The number of apoptotic neuronal cells in A group were different from them in both B and C group significantly. The number of apoptotic neuronal cells in C group is significant less than that in C group(p<0.01,P<0.05)4、ImmunohistochemistryThere were a few expression in A group. The expression of B and C group were mainly located in ischemic penumbra. The expression of bcl-2 and bax began to increase after 3h following cerebral ischemia/reperfusion, reached the peak at 24h, the peak of bcl-2 continuing to 72h following cerebral ischemia/reperfusion, the expression of bax began to decrease after 72h following cerebral ischemia. The expression of bcl-2 in C group is significantly enhanced than that in B group, the expression of bax in C group is weaker than that in B group.5、ElisaThere were a few IL-1βin A group. IL-1βbegan to increase after 3h following cerebral ischemia/reperfusion, reached the peak at 72h. the number of IL-1βin C group is less than that in B group significantly.6、Reverse transcription-polymerase chain reaction (RT-PCR)There were a little expression of caspase-3mRNA and caspase-8 mRNA. The expression of caspase-3mRNA and caspase-8 mRNA gradually increased as time went on following ischemia/reperfusion, reached the peak at 24h following cerebral ischemia/reperfusion. The expression of caspase-3mRNA and caspase-8 mRNA in D group is less than that in B group significantly.7、Electron-microscopeThe neurons、gliocyte and structure of capillaries were in integrity in A group. There were a lot of vacuole in neuron and organelle disappeared in B group. There were also a lot of vacuole in gliocyte and the cavity of capillaries became smaller, vascular endotheliocyte became bigger in B group. In C group there were a few vacuole and a lot of organelle such as endoplasmic reticulum、mitochondria、ribosomes, nucleus was big and chromatin was uniform in neuron; endoplasmic reticulum was expansion mildly, the number of golgi body became more in gliocyte; the cavity of capillaries became bigger and endotheliocyte became smooth.Conclusion:1、Acupuncturing the head point-line of anterior oblique line of vertex-teporal and posterior oblique line of vertex-teporal can improve the scores of neurological functions deficits, decrease the infarct volume and make the structure of neuron keeping integrity significantly. this result indicate that acupuncturing the head point have protective effect on cerebral ischemia/reperfusion.2、Acupuncturing the head point has the protective effect on cerebral ischemia/reperfusion by decreasing neuronal apoptosis in ischemia penumbra.3、Acupuncturing the head point can significantly increase bcl-2 and decrease bax 4、Acupuncturing the head point can reduce inflammation reaction, lower IL-1βof ischemic penumbra.5、Acupuncturing the head point can significantly decrease the expressions of caspase-3mRNA and caspase-8 mRNA following cerebral ischemia/reperfusion and inhibit the neuronal apoptosis.更多还原