【作者】 王渊；

【导师】 艾炳蔚；

【作者基本信息】 南京中医药大学 ， 针灸推拿学， 2013， 博士

【摘要】 目的针刺对机体的影响虽然是多方面的,但同一穴位是否对同一器官在不同的病理状态下存在双向调节作用,也就是在激发机体自身固有的调节系统的前提条件下,可以起到这种疗法防治疾病、增强机体免疫能力的作用,是本课题的主要研究目标。我们从功能性肠病切入,以功能性腹泻和功能性便秘这两种相反病理生理状态的疾病为研究对象,采用电针刺激可调节胃肠运动的穴位“天枢”、“大肠俞”、“曲池”、“上巨虚”,观察其对与大鼠胃肠运动相关的脑肠肽含量和c-kit蛋白表达的影响,研究不同穴位对同一内脏活动的“双向调节机制”,系统探讨电针刺激穴位引起双向调节效应的作用靶点以及促使这种效应产生的生物学机制。方法本论文分为理论研究和实验研究两部分。理论研究部分是通过有关针灸治疗功能性便秘(FC)、功能性腹泻(FD)的古今文献、现代研究进展回顾、挖掘、分析,预期证实针灸是功能性便秘和腹泻的重要治法。实验研究部分首先采用番泻叶灌胃复制FD大鼠模型,冰水灌胃复制FC大鼠模型,在造模结束后,采用ELISA法及免疫印迹动态观察模型大鼠血清及组织中GAS、SP、 Ghrelin、VIP、SS、GAL及结肠组织c-kit蛋白的动态表达,探讨其在FD和FC发病中的作用。其次是在成功复制FD和FC大鼠模型的基础上,采用电针刺激不同穴位组合对FD和FC大鼠进行干预治疗,观察其对FD和FC大鼠血清及组织中GAS、SP、Ghrelin、VIP、SS、GAL含量及结肠组织中c-kit蛋白表达的影响,从分子水平探讨电针双向调节功能性肠病的作用机理,探讨同一穴位是否对同一器官在不同的病理状态下存在双向调节作用的生物学机制。结果(1)采用番泻叶灌胃复制FD大鼠模型,模型组大鼠随着造模时间不同其体质量增长率、腹泻指数、稀便率、稀便级,与空白组比较,有显著性差异(P<0.01),模型组大鼠体质量呈负增长但趋势平缓,腹泻指数、稀便率、稀便级均较空白组显著性增多。模型大鼠结肠通过HE染色在光镜下可以呈现以下表现：结肠粘膜形态完好,粘膜下未现充血、水肿,上皮细胞排列整齐,尚无炎性细胞浸润。采用番泻叶灌胃复制FC大鼠模型,模型组大鼠随着造模时间不同其体质量增长率、24小时粪便粒数、24小时粪便质量、粪便含水量及首粒黑便排出时间,与空白组比较,有显著性差异(P<0.01),模型组大鼠体质量增长但趋势平缓,24小时粪便粒数、24小时粪便质量、粪便含水量均较空白组显著性减少,首粒黑便排出时间较空白组显著性增加。模型大鼠结肠通过HE染色在光镜下可以呈现以下表现：结肠粘膜形态完好,粘膜下未现充血、水肿,上皮细胞排列整齐,尚无炎性细胞浸润。(3)对于FD大鼠,与空白组比较,模型组均可使血清及组织中GAS、SP、 Ghrelin含量显著增高,经统计学处理,有显著性差异(P<0.01),同时模型组均可使血清及组织中VIP、SS、GAL含量显著降低,经统计学处理,有显著性差异(P<0.01)。对于FC大鼠,与空白组比较,模型组均可使血清及组织中GAS、SP、Ghrelin含量显著降低,经统计学处理,有显著性差异(P<0.01),同时模型组均可使血清及组织中VIP、SS、GAL含量显著性增高,经统计学处理,有显著性差异(P<0.01)。(4)对于FD大鼠,空白组相比,模型组大鼠c-kit的蛋白表达明显增加(P<0.01)。对于FC大鼠,空白组相比,模型组大鼠c-kit的蛋白表达明显减少(P<0.01)。(5)对于FD大鼠,在电针干预第11天,各电针治疗组均可使大鼠1小时内排便粒数、腹泻指数、稀便率和稀便级显著降低,与模型组比较差异有统计学意义(P<0.01),电针Ⅰ组对其改善最为明显。对于FC大鼠,在电针干预第11天,各电针治疗组均可使大鼠24小时粪便粒数、24小时粪便质量、粪便含水量显著增加,与模型组比较差异有统计学意义(P<0.01),电针Ⅱ组对其改善最为明显。(6)对于FD大鼠,与模型组比较,各电针治疗组均可使血清及绝大多数组织中GAS、SP、Ghrelin含量降低,VIP、SS、GAL含量升高,经统计学处理,有显著性差异(P<0.05或P<0.01),但均不能恢复到正常水平,电针Ⅰ组可使大鼠血清及绝大多数组织中GAS、SP、Ghrelin含量下降及VIP、SS、GAL含量升高并接近正常水平。对于FC大鼠,与模型组比较,各电针治疗组均可使血清及绝大多数组织中GAS、SP、Ghrelin含量升高,VIP、SS、GAI含量降低,经统计学处理,有显著性差异(P<0.05或P<0.01),但均不能恢复到正常水平,电针Ⅱ组可使大鼠血清及绝大多数组织中GAS、SP、Ghrelin含量升高,VIP、SS、GAL含量降低并接近正常水平。(7)对于FD大鼠,与模型组相比,电针治疗三组可以使c-kit的蛋白表达减少,电针1组c-kit的蛋白表达明显减少(P<0.05),电针Ⅱ组和电针Ⅲ组c-kit的蛋白表达减少不明显,经统计学处理,无显著性差异(P>0.05)。电针Ⅰ组与电针Ⅱ组、电针Ⅲ组相比c-kit的蛋白表达经统计学处理,有显著差异(P<0.05)。对于FC大鼠,与模型组相比,电针治疗三组c-kit的蛋白表达明显增加(P<0.05),电针Ⅱ组c-kit的蛋白表达明显增加(P<0.05),电针Ⅱ组与电针Ⅰ组、电针Ⅲ组相比c-kit的蛋白表达有显著差异(P<0.05)。结论(1)采用番泻叶灌胃可成功复制FD模型,冰水灌胃可成功复制FC模型,模型具有良好的稳定性。(2)脑肠肽GAS、SP、Ghrelin、VIP、SS、GAL和c-kit蛋白的平衡参与了FD和FC的发生、发展。(3)对于FD和FC模型大鼠,其结肠组织中c-kit蛋白的表达与脑肠肽GAS、 SP、Ghrelin的表达之间均存在程度相等的效应关系,呈正相关,与脑肠肽VIP、 SS、GAI的表达之间均存在程度不等的效应关系,呈负相关。(4)电针“天枢”、“大肠俞”、“曲池”、“上巨虚”对胃肠运动的调节作用均与相关脑肠肽含量及c-kit表达的变化有着密不可分的联系。其中“天枢”、“大肠俞”对胃肠功能发挥着一定的调节作用,这种调节作用主要表现为抑制胃肠运动,“曲池”、“上巨虚”对胃肠功能发挥着一定的调节作用,这种调节作用主要表现为促进胃肠运动。针刺上述穴位对胃运动的这种调节作用可能是通过激活脑肠肽对胃肠平滑肌细胞动力调节的信号转导通路以及调节c-kit蛋白表达而实现的。而且,在本研究中,穴位的双向调节作用并不是按照传统的共识表现为同一穴位组合对不同病理状态下的内脏器官存在着双向调节效应,而是表现为不同穴位的组合发挥综合的良性的调节作用。更多还原

【Abstract】 Objective Although acupuncture has multifold affectings on people, the same point plays the bidirectional regulatory role which means by triggering the body’s own natural regulation system to prevent and cure diseases, strengthen the immunefuncion on the same organ in different pathological state is still unclear. In this research, we use functional diarrhea and functional constipation those two opposing pathophy-siological states of the functional bowel as the object to study the bidirectional regulatory founction in gastrol systerm. we choose the gastrointestinal motility points Tianshu Dachangshu "Quchi on Shangjuxu, and use electro-acupuncture to stimulate them, and observe the rat of gastrointestinal motility, brain-gut peptide content and c-kit protein expression. Through this reaserch we hope to find if the different points on the same visceral activities" two-way adjustment mechanism, the system explore bi-regulating effect caused by the acupuncture points, and the biological mechanism of this effect.Method This essay contents theoretical and experimental parts. Theoretical research part includes the ancient and modern literature, modern research review, mining, analysis about the acupuncture treatment of functional constipation (FC), functional diarrhea (FD). The purpose is to confirm Acupuncture is an important method to cure functional constipation and diarrhea. Experimental research part is to explore the biological mechanisms of the bi-way adjustment, that whether or not the same point on the same organ has the bidirectional mechanisms in different pathological states. In this part, first, we use senna orally to copy FD rat model and ice water gavage to copy FC rat model. After modeling, we observe the GAS, the SP Ghrelin, VIP, SS, GAL and colon tissue c-kit protein expression dynamically by the meusuments of ELISA and Western blot to explore and its role in the FD and FC pathogenesis. Then, we use electro-acupuncture to stimulate differet acupoints-coalivations on the FD&FC rats we modeling to see if those indexes has been affected. Results (1) Compared with the control group, the growth rate of body mass, diarrhea index the loose stools rate, loose stools level of the FD rats which being modeled by using the senna gavage are significant differet (P<0.01). Compared with blank control group, the model group rats have lower body weight and higher diarrhea rate, loose stools rate, and loose stools level. Under the HE staining, the colon section of the modeling rats can be seen full of intact colonic mucosa and neat arranged epithelial cells without submucosal hyperemia, edema, infiltration of inflammatory cells.(2)Compared with the control group, the growth rate of body mass,24-hour fecal grains, the24-hour stool quality, stool water content and the first grain black discharge time rats of the FC rats which being modeled by using the ice water gavage are significant differet (P<0.01). Compared with blank control group, the model group rats have lower body weight,24-hour stool grains,24-hour stool quality, and fecal water content and longer. first grainmelena discharge time. Under the HE staining, the colon section of the modeling rats can be seen full of intact colonic mucosa and neat arranged epithelial cells without submucosal hyperemia, edema, infiltration of inflammatory cells.(3) Compared with the control group,the GAS, SP, Ghrelin contents in the serum and tissues of modeling FD rats elevated, and the VIP, SS, GAL contents decreased, and the difference was statistically significant (P<0.01).Compared with the control group, the GAS, SP, Ghrelin contents in the serum and tissues of modeling FC rats decreased, and the VIP, SS, GAL contents elevated, and the difference was statistically significant (P<0.01).(4) Compared with the blank group, the expression of the c-kit protein of the modeling FD rats increased, and the expression of the c-kit protein of the modeling FD rats decreased, and both difference were statistically significant (P<0.01).(5)Compared with model group, after11days of electro-acupuncture intervention, each group of the FD rats has lower defecation grains, diarrhea index, loose stools and loose stools level within one hour, and the difference was statistically significant (P<0.01). Among each group,electro-acupuncture group I has the most obvious improvement.Compared with model group, after11days of electro-acupuncture intervention, each group of the FC rats has higher feces grains rats24hours,24hours stool quality, and fecal water content, and the difference was statistically significant (P<0.01). Among each group,electro-acupuncture group II has the most obvious improvement.(6) Compared with the model group, the GAS、SP、Ghrelin contents increased and the VIP、SS、GAL contents decreased in each electro-acupuncture treatment group, and the differences were statistically significant (P<0.01).We could not restored those contents to normal levels, howerer. Those contents in group I are almost near normal levels after intervention.Compared with the model group, the GAS、SP、Ghrelin contents deceased and the VIP、SS、GAL contents increased in each electro-acupuncture treatment group, and the differences were statistically significant (P<0.01).We could not restored those contents to normal levels, howerer. Those contents in group Ⅱare almost near normal levels after intervention.(7) Compared with the model group, the protein expression of c-kit the FD rats reduced in each electro-acupuncture treatment group, and was significantly decreased (P<0.05) in group I. The other two groups (Ⅱ、Ⅲ) have not show this significance(P>0.05).Compared with the group(Ⅱ、Ⅲ)the decrease of protein expression of c-kit in group I was significantly different (P<0.05).Compared with the model group, the protein expression of c-kit of the FC rats increased in each electro-acupuncture treatment group, and the diference was significant (P<0.05) in all the three groups. Compared with the group(Ⅰ、Ⅲ)the increase of protein expression of c-kit in group II was significantly different (P <0.05).Conclusion (1)Senna gavaging can replicate the FD model successfully, and ice water gavaging can replicate the FC model successfully. Both models have good stability. (2) The balance of brain-gut peptide includ GAS, SP, Ghrelin, VIP, SS, the GAL and c-kit protein involved in both the occurrence and the development of FD and FC.(3) For the FD and FC model rats, the expression of c-kit protein in colon tissue has the equal degree of effect relationship with the brain-gut peptide GAS, SP, Ghrelin expression,and the correlate relationship was positive. The expression of c-kit protein in colon tissue has the varying degree of effect relationship with the brain-gut peptide SS, GAL expression,and the correlate relationship was egative.(4) Using electric needle to stimulus "Tianshu"、"Dachangshu "、" Quchi"、"Shangjuxu" can cause the changes of the brain-gut peptide content and the expression of c-kit, which are closely related with Regulation of gastrointestinal function. Among those four acupoints, Tianshu and Dachangshu have the function to inhibit gastrointestinal motility, the other tows can promote gastrointestinal motility. Using electric needle to stimulus "Tianshu"、"Dachangshu"、" Quchi"、"Shangjuxu" can accommodate gastrointestinal motility, and such regulation was related to activate sympathetic and parasympathetic of digestive apparatus directly or indirectly. The adjustment function of electroacupuncture stimulating on these acupoints may rely on activating the signal transduction access of gastrointestinal smooth muscle cell dynamic adjustment triggered by braingut petide and adjusting the expression of c-kit protein.In this research,the two-way adjustment function on the internal organs under different pathological states has not being elicit by stimulating the single acupoint but by combinating the different acupoints. This conclution was not consist with the past opinion.更多还原