脾气虚证大鼠小肠肠神经-Cajal间质细胞-平滑肌网络损伤和针刺足三里、天枢穴修复作用的实验研究

发布时间：2018-05-24 04:25

本文选题：脾气虚证 + 针刺　；参考：《大连医科大学》2017年博士论文

【摘要】：背景:脾气虚证(Syndrome of Deficiency of Spleen-Qi,SDSQ)是临床上最常见的虚证之一,临床上主要是以进餐后胃部饱胀不适、消瘦乏力、倦怠易疲劳、面色枯黄无血色、大便稀溏等为临床表现。现代西方医学研究证实:脾气虚证的主要表现是胃肠道运动功能、消化吸收功能、内分泌功能和免疫功能等多系统、多因素的紊乱。然而消化道运动功能的恢复是消化吸收功能、内分泌和免疫功能低下等系统恢复的前提条件。胃肠道运动功能的恢复既是纠正胃肠道损害的最初始同时也是最关键的一步。促进胃肠道动力的治疗能够有效的治疗胃肠道运动功能、消化吸收功能、内分泌和免疫功能障碍等。所以说,探讨脾气虚证胃肠道运动功能障碍的本质,研究治疗脾气虚证胃肠道运动功能障碍的有效方法变得十分必要。针灸作为祖国医学中一种传统的治疗方法,在中国应用已有两千多年的历史。在临床实践中,越来越多的研究显示,针刺足三里、天枢穴可以纠正胃肠道运动功能紊乱,有效的治疗脾气虚证,在治疗功能性胃肠道疾病方面有着自己独特的优势,但确切的机制仍不十分清楚,需要进一步的研究。目的:通过苦寒泻下结合耗气破气的方法建造脾气虚证大鼠模型,以脾气虚证大鼠小肠肠神经-Cajal间质细胞-平滑肌细胞网络为研究对象,观察脾气虚证大鼠小肠肠神经-Cajal间质细胞-平滑肌细胞网络的损伤,研究脾气虚证的发病机制。然后对脾气虚证大鼠进行针刺足三里、天枢穴进行治疗,观察针灸对脾气虚证大鼠小肠肠神经-Cajal间质细胞-平滑肌细胞网络损伤的修复作用,进一步探索针刺足三里、天枢穴治疗脾气虚证的作用机制。方法:选取清洁级成年Wistar大鼠,雌雄不限,参考中国中医科学院陈小野教授的方法,先通过体质筛选,再采用苦寒泻下结合耗气破气的方法建造脾气虚证大鼠动物模型。将大鼠分为对照组、模型组、自然恢复组、假针刺穴位组(针刺非经非络穴位组)、针刺天枢穴治疗组、针刺足三里穴治疗组、针刺足三里+天枢穴联合治疗组。造模成功及治疗结束后,采用颈椎脱臼法处死大鼠,打开腹腔切取小肠的近幽门端用于实验。本实验研究分为以下三部分:第一部分、通过观察一般行为学变化、监测体重、测定尿d-木糖排泄率、大体解剖观察、测定胃排空率、测定小肠推进率、病理学he染色观察、透射电镜超微结构检测、免疫荧光标记激光扫描共聚焦显微镜检测等方法来观察脾气虚证大鼠小肠肠神经-icc-平滑肌网络的形态学变化和针刺足三里、天枢穴的修复作用。第二部分、通过免疫荧光双重标记激光扫描共聚焦显微镜检测的方法来观察脾气虚证大鼠小肠肠神经-icc之间的信号传导通路的损伤和针刺足三里、天枢穴的修复作用。第三部分、通过免疫组织化学、westernblotting、qpcr等方法来观察脾气虚证大鼠小肠icc-平滑肌之间的信号传导通路的损伤和针刺足三里、天枢穴的修复作用。通过三部分实验来探索脾气虚证所导致的胃肠道运动功能障碍的实质和针刺足三里、天枢穴治疗脾气虚证的作用机制。结果:1.与正常对照组相比,脾气虚证模型组大鼠的一般行为学表现明显变差,大体解剖肉眼观察小肠蠕动明显缓慢,小肠组织he病理学表现小肠组织结构明显破坏,icc的超微结构遭到严重破坏,体重增长明显缓慢,尿d-木糖排泄率、胃排空率、小肠推进率均明显降低,肠神经系统的pgp9.5神经纤维、ach神经纤维、sp神经纤维、vip神经纤维、no神经纤维、icc、smc、ens-icc-smc网络的数量明显减少,结构遭到明显破坏(p0.05),而模型组、自然恢复组、假针刺组三组之间相互比较,无明显差异(p0.05)。与模型组相比,天枢组、足三里组略有好转(p0.05),但天枢组、足三里组两组之间比较,无明显差异(p0.05)。与天枢组、足三里组相比,联合治疗组效果明显好转(p0.05),但仍低于正常对照组(p0.05)。总体来说,其基本趋势为:模型组≈自然恢复组≈假针刺组天枢组≈足三里组联合组正常组。2.与正常对照组相比,脾气虚证模型组大鼠小肠肠神经-icc之间的信号传导通路,主要有ach神经纤维的m受体、sp神经纤维的nk受体、no神经纤维的受体-鸟苷酸环化酶(sgc)、vip神经纤维的vpac受体、pka、pkc、cgk/pkg数量明显减少,其信号通路g蛋白偶联受体-camp-pka途径、g蛋白偶联受体-磷脂酶c(plc)-pkc途径、sgc-cgmp-pkg途径明显遭到破坏(p0.05),而模型组、自然恢复组、假针刺组三组之间相互比较,无明显差异(p0.05)。与模型组相比,天枢组、足三里组略有好转(p0.05),但天枢组、足三里组两组之间比较,无明显差异(p0.05)。与天枢组、足三里组相比,联合治疗组效果明显好转(p0.05),但仍低于正常对照组(p0.05)。总体来说,其基本趋势为:模型组≈自然恢复组≈假针刺组天枢组≈足三里组联合组正常组。3.与正常对照组相比,脾气虚证模型组大鼠小肠icc-平滑肌之间的信号传导通路,主要是缝隙连接和缝隙连接蛋白43数量明显减少,信号传导途径明显破坏(p0.05),而模型组、自然恢复组、假针刺组三组之间相互比较,无明显差异(p0.05)。与模型组相比,天枢组、足三里组略有好转(p0.05),但天枢组、足三里组两组之间比较,无明显差异(p0.05)。与天枢组、足三里组相比,联合治疗组效果明显好转(p0.05),但仍低于正常对照组(p0.05)。总体来说,其基本趋势为:模型组≈自然恢复组≈假针刺组天枢组≈足三里组联合组正常组。结论:1.脾气虚证时,大鼠一般行为学表现明显变差,体重增长缓慢,尿d-木糖排泄率明显降低,大体解剖观察小肠蠕动明显缓慢,胃排空率明显降低,小肠推进率明显降低,小肠病理学he染色观察小肠组织结构明显破坏,icc超微结构明显破坏,小肠的pgp9.5神经纤维、ach神经纤维、sp神经纤维、no神经纤维、vip神经纤维、icc、smc数量明显减少,网络结构明显破坏。针刺足三里和天枢穴治疗后,可明显修复脾气虚证的损伤,其基本趋势为:模型组≈自然恢复组≈假针刺组天枢组≈足三里组联合组正常组。2.脾气虚证时,大鼠小肠肠神经-icc之间的信号传导通路,主要有ach神经纤维的m受体、sp神经纤维的nk受体、no神经纤维的受体-鸟苷酸环化酶(sgc)、vip神经纤维的vpac受体、pka、pkc、cgk/pkg数量明显减少,其信号通路g蛋白偶联受体-camp-pka途径、g蛋白偶联受体-磷脂酶c(plc)-pkc途径、sgc-cgmp-pkg途径明显破坏。针刺足三里和天枢穴治疗后,可明显修复脾气虚证的损伤,其基本趋势为:模型组≈自然恢复组≈假针刺组天枢组≈足三里组联合组正常组。3.脾气虚证时,大鼠小肠ICC-平滑肌之间的信号传导通路-缝隙连接、缝隙连接蛋白43的数量明显减少,信号传导途径明显破坏。针刺足三里和天枢穴治疗后,可明显修复脾气虚证的损伤,其基本趋势为:模型组≈自然恢复组≈假针刺组天枢组≈足三里组联合组正常组。
[Abstract]:Background: spleen qi deficiency syndrome (Syndrome of Deficiency of Spleen-Qi, SDSQ) is one of the most common deficiency syndrome in clinical. The main clinical manifestations are stomach distention, fatigue, fatigue, dull complexion, loose stool and so on. Modern western medical research confirms that the main manifestation of spleen qi deficiency syndrome is gastrointestinal tract. Movement function, digestion and absorption function, endocrine function and immune function and many other systems, multiple factors disorder. However, the recovery of digestive tract motor function is the precondition for the recovery of digestive and absorption functions, endocrine and immune function. The recovery of gastrointestinal motility is both the initial and the most important to correct gastrointestinal damage. The key step is to promote the treatment of gastrointestinal motility, which can effectively treat gastrointestinal motility, digestion and absorption, endocrine and immune dysfunction. Therefore, it is necessary to explore the essence of gastrointestinal motility dysfunction in spleen qi deficiency syndrome and to study the effective methods of treating gastrointestinal motility dysfunction in spleen qi deficiency syndrome. As a traditional treatment method in Chinese medicine, it has been used in China for more than two thousand years. In clinical practice, more and more studies have shown that acupuncture at Zusanli, Tianshu point can correct gastrointestinal motility disorders, effectively treat spleen qi deficiency syndrome, and have its own unique advantages in the treatment of functional gastrointestinal diseases. But the exact mechanism is still not very clear, and further study is needed. Objective: to build a rat model of spleen qi deficiency syndrome by using the method of bitter cold and diarrhea combined with gas consumption and gas breaking, taking the mesenteric smooth muscle cell network of the intestinal nerve -Cajal as the research object in the spleen Qi deficiency rats, and observing the -Cajal interstitial cells of the intestinal nerve of the small intestine of the spleen qi deficiency rats. The mechanism of smooth muscle cell network was studied and the pathogenesis of spleen qi deficiency syndrome was studied. Then the rats of spleen qi deficiency syndrome were treated with acupuncture at Zusanli and Tianshu acupoint for treatment. The repair effect of acupuncture on the damage of mesenteric -Cajal stromal cells - smooth muscle cell network in the intestinal nerve of rats with spleen qi deficiency syndrome was observed, and the acupuncture at Zusanli and Tianshu point were used to treat the deficiency of Spleen Qi. Methods: to select the clean adult Wistar rats, the male and female rats are not limited. Referring to the methods of Professor Chen Xiaoye of Chinese Academy of Chinese medicine, the rat model of spleen qi deficiency syndrome is constructed by physical screening, and then using the method of bitter cold diarrhea and gas consumption breaking gas. The rats are divided into the control group, the model group, the natural recovery group and the false acupuncture point. Group (acupuncture non meridian acupoint group), acupuncture Tianshu acupoint treatment group, acupuncture Zusanli treatment group, acupuncture Zusanli + Tianshu point combined treatment group. After the success of the model and the end of the treatment, the cervical dislocated method was used to kill the rats and open the intraperitoneal incision of the proximal pylorus for the actual test. The experimental study was divided into the following three parts: the first part After observing general behavioral changes, monitoring body weight, measuring urine d- xylose excretion rate, gross anatomical observation, measuring gastric emptying rate, measuring small intestine propulsion rate, pathological HE staining, ultrastructural examination of transmission electron microscope, and immunofluorescent laser scanning confocal microscopy to observe -icc- level of intestinal nerve in small intestine of rats with Spleen Qi deficiency syndrome The morphological changes of smooth muscle network and the effect of acupuncture on Zusanli and Tianshu point. The second part is to observe the damage of the signal transduction pathway between the -icc of the intestinal nerve in the small intestine of rats with spleen qi deficiency syndrome by the method of double immunofluorescence double labeling laser scanning confocal microscopy, and the repair effect of the acupuncture point in the Zusanli and the Tianshu point. Third parts. By means of immunohistochemistry, westernblotting, qPCR and other methods to observe the damage of the signal transduction pathway between icc- smooth muscle in the small intestine of spleen qi deficiency rats and the repair effect of acupuncture at Zusanli and Tianshu point. The essence of the gastrointestinal motility dysfunction caused by spleen qi deficiency syndrome and the acupuncture at Zusanli, Tianshu and Tianshu are explored by three parts of experiments. Results: 1. compared with the normal control group, the general behavior of the rats in the model group of spleen qi deficiency syndrome was significantly worse than that in the normal control group, and the small intestinal peristalsis was obviously slowed down by the gross anatomy of the naked eye. The he pathology of the small intestine showed the obvious destruction of the small intestine structure, the ultrastructure of the ICC was seriously damaged and the weight increased obviously. Slow, urinary d- xylose excretion rate, gastric emptying rate and small intestinal propulsion rate decreased significantly. The number of PGP9.5 nerve fiber, ACh nerve fiber, SP nerve fiber, VIP nerve fiber, no nerve fiber, ICC, SMC, ens-icc-smc network in the intestinal nervous system decreased significantly, and the structure was obviously destroyed (P0.05), while the model group, natural recovery group, and false acupuncture group were three groups. Compared with the model group, there was a slight improvement in the Tianshu group and Zusanli group (P0.05), but there was no significant difference between the two groups in the Tianshu group and the Zusanli group (P0.05). Compared with the Tianshu group, the effect of the combined treatment group was better than the Zusanli group (P0.05), but it was still lower than that of the normal control group (P0.05). Overall, the base of the group was the base of the group (P0.05). Compared with the normal control group, the normal group.2. of the Zusanli group of the Zusanli group was compared with the normal control group, and the signal transduction pathway between the -icc of the intestinal and intestinal nerve in the model group of spleen qi deficiency syndrome group was mainly the M receptor of ACh nerve fiber, the NK receptor of SP nerve fiber, the receptor of no nerve fiber receptor cyclization of guanosine acid. The number of VPAC receptors, PKA, PKC, cgk/pkg decreased significantly in SGC, PKA, PKC, cgk/pkg, G protein coupled receptor -camp-pka pathway, G protein coupled receptor phospholipase C (PLC) -pkc pathway, and the model group, natural recovery group and false acupuncture group were compared with each other, and there was no significant difference. Compared with the model group, Tianshu group and Zusanli group had a slight improvement (P0.05), but there was no significant difference between the two groups in the Tianshu group and the Zusanli group (P0.05). Compared with the Tianshu group and Zusanli group, the effect of the combined treatment group was obviously improved (P0.05), but still lower than the normal group (P0.05). Compared with the normal control group, the signal transduction pathway between the icc- smooth muscle of the small intestine of the model group of spleen qi deficiency syndrome group and the normal group of the combination group of the Zusanli group of the Tianshu group of the sham acupuncture group, the signal transduction pathway of the gap junction and the gap junction protein 43 was significantly reduced, the signal transduction pathway was obviously broken (P0.05), and the model group, the natural recovery group, and the false acupuncture group were used in the model group of spleen qi deficiency syndrome group. There was no significant difference between the three groups (P0.05). Compared with the model group, the Tianshu group and Zusanli group had a slight improvement (P0.05), but there was no significant difference between the Tianshu group and the Zusanli group (P0.05). Compared with the Tianshu group and Zusanli group, the effect of the combined treatment group was obviously improved (P0.05), but still lower than that of the normal control group (P0.05). Conclusion: 1. when spleen qi deficiency syndrome, the general behavior of the rats was significantly worse, the body weight increased slowly, the urine d- xylose excretion rate decreased significantly, the gross anatomic observation of the small intestinal peristalsis was slow, the gastric emptying rate was significantly reduced and the small intestine was pushed. The small intestinal pathology HE staining showed that the structure of small intestine was obviously destroyed and the ultrastructure of ICC was obviously destroyed. The PGP9.5 nerve fibers of the small intestine, ACh nerve fiber, SP nerve fiber, no nerve fiber, VIP nerve fiber, ICC, SMC were obviously reduced, and the network structure was obviously destroyed. The basic trend of the syndrome of qi deficiency syndrome of the spleen qi deficiency syndrome was as follows: the signal transduction pathway between the -icc of the intestinal nerve of the small intestine in the normal group of the combination group of the Zusanli group of the Zusanli group and the normal group of the Zusanli group was.2., mainly including the M receptor of the ACh nerve fiber, the NK receptor of the SP nerve fiber, the receptor of the no nerve fiber receptor - guanosine ring. SGC, the number of VPAC receptors, PKA, PKC, cgk/pkg in VIP nerve fibers obviously decreased. The signal pathway of G protein coupled receptor -camp-pka pathway, G protein coupled receptor phospholipase C (PLC) -pkc pathway was significantly destroyed. After acupuncture at Zusanli and Tianshu acupoints, the damage of spleen qi deficiency syndrome could be obviously repaired. The basic trend was the model. The signal transduction pathway - gap junction between the ICC- smooth muscle of the small intestine of the rat's small intestine, the number of gap junction protein 43 decreased obviously, and the signal transduction pathway was obviously destroyed. After the acupuncture foot three li and Tianshu acupoint treatment, the spleen qi could be obviously repaired after the treatment of the acupuncture at the point of.3.. The basic trend of deficiency syndrome was: model group, natural recovery group, pseudo acupuncture group, Tianshu group, Zusanli group and normal group.
【学位授予单位】：大连医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R245

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