【作者】 卓缘圆；

【导师】 杨卓欣；

【作者基本信息】 广州中医药大学 ， 针灸推拿学， 2010， 博士

【摘要】 近年来中医对脾虚证进行了大量有价值的探索性研究工作,脾虚证不仅对消化系统、免疫功能等方面带来损害,还对中枢神经系统造成影响,如脾虚可引起脑内神经元、神经递质、神经营养因子、信号转导、基因表达等相关的改变,还可以影响脑神经细胞的分化和增殖,改变组织的重塑能力,从而引起皮质和海马等脑组织的结构的改变,进而影响脑功能。针刺疗法在脾虚证的治疗中有很好的疗效,具有一定的临床和实验基础,因而我们设想,对于脾虚证引起的脑神经细胞损害,是否能够通过纠正脾虚证,改善脾虚的状态,从而有效地保护损伤的神经细胞,促进脑神经功能的恢复。基于此种设想及参阅大量文献,本课题以神经干细胞的增殖和分化为切入点,从文献研究与实验研究两方面,探讨电针治疗对脾虚证引起神经细胞损伤的保护作用和机制。1、目的：观察脾虚证幼鼠侧脑室下区、海马齿状回区的细胞结构和神经干细胞增殖、分化情况,同时观察这两个脑区碱性成纤维细胞生长因子(bFGF)及其mRNA的表达,以及电针足三里、三阴交穴位对细胞结构、神经干细胞增殖、分化和碱性成纤维细胞生长因子(bFGF)及其mRNA表达的影响,以期从神经干细胞角度揭示电针对脾虚证幼鼠中枢神经保护作用的途径和机制,为针灸治疗脾虚证引起的中枢神经细胞损伤提供理论和实验依据。2、方法：SPF级幼年健康雄性SD大鼠192只,鼠龄为4周龄,体重55±5g。适应环境饲养3天后开始实验。按随机数字表法分为正常组、模型组、电针组,每组64只。每组再分别随机分为7天组、14天组、28天组和49天组四个亚组,每亚组16只。采用大黄灌胃法、利血平腹腔注射法联合饥饱失常法制作实验性大鼠脾虚证模型,造模时间为14天,观察脾虚证大鼠的外观表现和体重变化,电针组在造模期结束后于双侧足三里、三阴交穴行电针治疗,采用疏密波刺激(密波15Hz),强度6-15V,持续时间为20分钟,每日一次至动物处死。模型组在造模期结束后固定于针刺操作台上20分钟,不作任何治疗。2.1各亚组随机选择2只大鼠,共24只大鼠分别在实验设定的时间点处死,进行灌注固定取脑,用光学显微镜和电子显微镜观察正常组、模型组和电针组大鼠侧脑室下区和海马齿状回区的病理形态学改变。2.2各亚组随机选择6只大鼠,共72只大鼠,分别在实验设定的时间点前两天进行Brdu腹腔注射,处死后进行灌注固定取脑,运用双重免疫组化方法对正常组、模型组和电针组大鼠侧脑室下区和海马齿状回区Brdu细胞、Brdu/Nestin. Brdu/GFAP和Brdu/NSE细胞进行标记,采用美国Image-Pro Plus专业图像分析软件进行图像分析,400倍光镜下进行阳性细胞计数,最后用Statal0.0软件进行统计分析,比较各组差异。2.3各亚组随机选择8只大鼠,共96只大鼠分别在实验设定的时间点处死,运用免疫组化法对正常组、模型组和电针组大鼠侧脑室下区和海马齿状回区的bFGF蛋白进行标记,采用美国Image-Pro Plus专业图像分析软件进行图像分析,200倍光镜下进行阳性细胞计数,并运用RT-PCR技术测定bFGF mRNA表达,最后用Statal0.0软件进行统计分析,比较各组差异。3、结果：3.1模型大鼠较正常大鼠明显消瘦,毛色不荣,并出现成群蜷缩、扎堆、拱背、精神倦怠、嗜卧、眼眯、四肢无力或见颤抖、反应迟钝、拉尾排便次数增多。3.2与正常组比较,光镜下观察模型组脑组织内神经元轮廓模糊,胞体肿大,周围间隙增宽,核碎裂,胞浆出现空洞,空泡形成,细胞带层次减少。电针组脑组织内神经元轮廓尚清晰,细胞核形态正常,细胞带尚均匀,可见有胶质细胞浸润。电镜下观察模型组脑结构在神经元胞体和神经毡区都有改变,尤以线粒体和突触终末端的改变明显,电针组神经元损伤程度较轻,细胞结构良好。3.3无论是在侧脑室下区或海马齿状回区,双重免疫组化都有相似的结果。正常组大鼠Brdu、Brdu/Nestin、Brdu/GFAP、Brdu/NSE的表达在各时间段均无统计学意义(P>0.05)。Brdu的表达在模型组7天组和14天组较正常组低(P<0.05),到28天组恢复到正常组水平；在电针组7天组表达已达正常水平,14天组的表达最为显著,在28天组和49天组逐渐减少,但仍多于同期正常组和模型组(P<0.05)。Brdu/Nestin阳性细胞的表达在模型组7天组仅有少量,在14天组达到正常水平(P>0.05),28天组达到高峰(P<0.05)；在电针组7天组即可与正常组相当,在14天组达到高峰值,28天和49天组逐渐减少,但与同期的模型组与正常组比较仍有统计学意义(P<0.05)。Brdu/GFAP阳性细胞的表达在模型组7天组和14天组均较同期正常组少(P<0.05),28天组可达到正常组水平(P>0.05)；而在电针组7天组即可达到正常的水平(P>0.05),14天组为高峰期,28天后逐渐减少,恢复到基线水平。Brdu/NSE阳性细胞的表达在模型组7天组和14天组均较同期正常组少(P<0.05),28天组可达到正常组水平(P>0.05)；而在电针组7天组即可达到正常的水平(P>0.05),14天组开始增多,28天组达到高峰值,以后逐渐恢复至正常水平。3.4无论是在侧脑室下区或海马区,bFGF及其mRNA在各时间段同期表达都是一致的。模型组bFGF及其mRNA表达在7天组处于基线水平,14天组明显增多,与同期正常组相比差异有非常显著性意义(P<0.05)；28天组仍停留在一个高水平,至49天回落,与正常组比较无统计学意义(P>0.05)。与模型组不同,电针组bFGF及其mRNA表达在7天组即处于较高的表达,与模型组和正常组比较具有显著性差异(P<0.01),14天组稍有下降,但较同期正常组仍有明显差异(P<0.01),并且这一表达水平可以持续至49天。4、结论本研究选择大黄灌胃、利血平腹腔注射联合饥饱失常法造成幼鼠实验性脾虚状态的病理模型,应用组织形态学、免疫组织化学等方法,围绕着神经干细胞的生长、增殖和分化对脑神经细胞的影响展开探讨,观察脾虚对脑神经细胞所造成的影响,同时分析电针对脾虚造成的神经细胞损害的保护作用及其机制。结果如下：脾虚证可出现脑神经元胞体和突触的改变,同时导致神经干细胞增殖和分化能力降低。电针“足三里”“三阴交”穴对脾虚所造成脑组织的损害有保护作用,使脑神经元损伤程度较轻,维持正常的细胞形态,保护神经毡中突触的基本结构,同时电针能促进脾虚证大鼠神经干细胞的增殖,适当地促进侧脑室下区和海马齿状回区增殖的神经干细胞往星形胶质细胞及神经元方向分化,电针的保护作用可能与促进bFGF蛋白及其mRNA表达上调有关。综上所述,我们认为对于脾虚证引起的中枢神经细胞损害,通过电针的刺激作用可以改善机体脾虚的功能状态,消除致病因素,并且诱导神经干细胞增殖、分化,从而使脾虚证所造成的受损的神经细胞得到修复。更多还原

【Abstract】 In recent years, there have been a great deal of valuable researches on the splenasthenic syndrome in the area of traditional Chinese medicine. Spleen deficiency refers not only to the functional disorder of digestive systems and immunological function, but also the related changes in brain neurotransmitters, neurotrophic factors, signal transduction, gene expression. What’s more, spleen deficiency can lead to the changes in the structure of cerebral cortex, hippocampus and other organizations by affecting nerve cell differentiation and proliferation, and changing the ability of organism’s remodeling, thereby affecting brain functions. There is a lot of clinical and experimental foundation to confirm that acupuncture can cure the splenasthenic syndrome. Therefore, for the damage to brain functions caused by splenasthenic syndrome, efforts are made in our researches to the recovery of brain functions through curing this syndrome or improving the splenic deficiency condition. Based on the assumption and literature study, we chose the differentiation and proliferation of nerve cell as cut-in point. From the aspects of literature and experiment study, we can approach the possible mechanism of electro-acupuncture on protecting nerve cell from damaging caused by splenasthenic syndrome.1. Objective:To observe the differentiation and proliferation of nerve stem cell and expression of bFGF protein and mRNA in subventricular zone(SVZ)and Denatate Gyrus(DG) in spleen deficiency Rats, and approach the effects of electro-acupuncturing Zusanli (ST36) and Sanyinjiao (SP6) on the differentiation and proliferation of nerve stem cell and expression of bFGF protein and mRNA. By observing nerve stem cell, we hope to find the possible mechanism of electro-acupuncture on protecting nerve cell from damaging caused by splenasthenic syndrome, and provide theoretical and experimental evidence for electro-acupuncture to treat nerve cell damage caused by splenasthenic syndrome.2. Methods:A total of 192 healthy, male, Sprague Dawley rats, weighing (55±5) g and aged 4 weeks, from SPF-grade laboratory were taken into the study. Before the test, they were raised to adapt to the surroundings. According to the random digits table, they were divided into control group, model group and electro-acupuncture treated group, each group contained 64 rats. Then each group was averagely divided into 7 days group,14 days group and 28 days group at random. There were 16 rats in each subset group. Spleen deficiency was induced via intragastric administration of rhubarb、intraperitoneal injection of reserpine connected with feeding irregular diet for 14 days. We observed the change of appearance and weight in the spleen deficiency rats. Rats in electro-acupuncture treated group were treated with electro-acupuncturing Zusanli and Sanyinjiao points after modeling. As for electric acupuncture, distant-dense wave was chosen. The frequency was15Hz for dense wave and intensity was 6 to15 V. The treatment lasted for twenty minutes, and was done once every day until the rats got killed. On the other hand, rats in model group were fixed on the operation board without any treatment for twenty minutes after modeling.2.1 We select 2 rats from each subset group randomly. All together 24 rats were killed at the design time, and their brains were taken after fixed by perfusion. Then we observed the pathomorphism of subventricular zone (SVZ) and Denatate Gyrus(DG) in normal group, model group and electro-acupuncture treated group by light microscope and electronmicroscope.2.2 We select 6 rats from each subset group randomly. All together 72 rats were injected Brdu peritoneal 2 days before the design time that they were killed, and their brains were taken after fixed by perfusion. Then we observed the cell marked by Brdu、Brdu/Nestin、Brdu/GFAP and Brdu/NSE in SVZ and DG in normal group, model group and electro-acupuncture treated group by immunohistochemistry. At last, we counted positive cells at light microscope of 400X, analyzed the image by analytical system of Image-Pro Plus software from USA, and compared the difference between groups by Stata 10.0 software.2.3 We select 8 rats from each subset group randomly. All together 96 rats were killed at the design time, and their brains were taken immediately. Immunohistochemistry and PCR reaction was performed to detect the protein and mRNA expression of bFGF in SVZ and DG. At last, we counted positive cells at light microscope of 200X, analyzed the image by analytical system of Image-Pro Plus software from USA, and compared the difference between groups by Stata 10.0 software.3. Results:3.1 Compared to the normal group, rats in the model group were obviously emaciated, poor hair quality, and rolling up together, lassitude, closing eyes, fatigue, reacting slowly, defecating frequently.3.2 Compared to the normal group at light microscope, the outline of neuron was blurred, the cell body was swelling, the interspace around cells was widen, and nuclear fragmentation, cavity in kytoplasm, besides, the zonal layer of cells was diminished in model group. But in electro-acupuncture treated group, the outline of neuron was clear, the cell nucleus was eumorphism, the zonal layer of cells was well-distributed, and the glial cells were infiltrating. At electron microscope, some structures of the pericaryon and neuropil were destroyed in model group, especially the structure of mitochondrion and the terminal of synapse. But in electro-acupuncture group, degree of injury in nerve cells is lower and most of the cell organs still exist.3.3 The same conclusion could be seen in the same period either in subventricular zone or Denatate Gyrus. The number of positive cells of Brdu、Brdu/Nestin、Brdu/GFAP、Brdu/NSE were coincident in different periods in the normal control group (P>0.05). The number of Brdu cells in model group was less than that in the normal group in the 7th day and the 14th day (P<0.05), but it recovered to the normal level in the 28th day. The number of Brdu cells in electro-acupuncture treated group was almost the same as that in the normal group in the 7th day, and it was extremely high in the 14th day, but it decreased in the 28th day and 49th day, however, the level was still higher than that in model group and normal group in this period (P<0.05). In model group, there was little Brdu/Nestin cells in the 7th day, while the number increased and reached to a normal level in the 14th day (P>0.05), then it achieved the peak level in the 28th day (P<0.05). In electro-acupuncture treated group, the number of Brdu/Nestin cells was just the same as that in normal group in the 7th day. And it reached to a peak level in the 14th day. In the 28th day and 49th day, the number declined but it was still higher than that in model group and normal group in this period (P<0.05). In model group, the number of Brdu/GFAP cells was less than that in normal group in the 7th day and the 14th day (P<0.05). But it reached to the normal level in the 28th day (P>0.05). The number of Brdu/GFAP cells in electro-acupuncture treated group was just the same as that in normal group in the 7th day (P>0.05). And it reached to a peak level in the 14th day. Then it delined to the basal line after the 28th day. The number of Brdu/NSE cells in model group was less than that in normal group in the 7th day and the 14th day (P<0.05), but it recovered to the normal level in the 28th day (P>0.05). In electro-acupuncture treated group, the number was just the same as that in normal group in the 7th day (P>0.05). And it increased in the 14th day and reached to a peak level in the 28th day, then delinced to the basic line gradually.3.4 The expression of bFGF protein and mRNA are coincident in the same period either in subventricular zone or Denatate Gyrus. In model group, the expression was low in the 7th day after model treated period. However, it increased quickly in the 14th day and the level was higher than that in normal group (P<0.05) It stayed for some time and delined to the basal line in the 49th day. The expression of bFGF protein and mRNA in electro-acupuncture treated group was different. It generally increased in the 7th day, and the level was higher than that in model group and in normal group (P<0.01). Then it slightly descended in the 14th day; however, the level was still higher than that in normal group in this period. The high level lasted to the 49th day.4. Conclusions:In this empirical study, we set up a spleen deficiency rat model by combinding with intragastric administration of rhubarb, intraperitoneal injection of reserpine and fed on irregular die. In order to observe the damage of nerve cell caused by splenasthenic syndrome, and provide theoretical and experimental evidence for electro-acupuncture protecting nerve cell, we observed the growth, differentiation and proliferation of nerve stem cell by histomorphology and immunohistochemistry. The results were as follows:The splenasthenic syndrome may result in the changes of pericaryon and synapses, and degrade the proliferation and differentiation potency of nerve stem cells. The electro-acupuncturing Zusanli and Sanyinjiao points treatment can inhibit the damage and protect the nerve cells. It maintains the normal structure of cells and synapse. Besides, it can enhance the proliferation potency of nerve stem cell in the spenasthenic syndrome, and encourage nerve stem cells differentiating to astrocyte and nerve cell in the Subventricular Zone of the Lateral Ventricle and dentate gyrus of hippocampus. The protection of electro-acupuncture may be relevant to maintaining a high level of bFGF and mRNA expression。In a word, we could consider that the encephalodysplasia due to the splenasthenic syndrome may be cured by electro-acupuncture. Besides, the elecrto-acupuncture can induce the cell proliferation and differentiation, so as to repair the nerve cells which are impaired by splenic asthenia.更多还原