【作者】 蒋峰；

【导师】 陈玉根；

【作者基本信息】 南京中医药大学 ， 中医外科学（学术型）， 2018， 博士

【摘要】 慢性便秘全球发病率约为14%,其中慢传输型便秘(STC)占慢性特发性便秘4种亚型之首,其主要特点是结肠运输缓慢、大便通过时间延长,伴或者不伴有腹胀、大便干燥。研究表明,结肠组织中Cajal间质细胞(ICC)数量的减少、水通道蛋白表达的异常在STC的病理过程中发挥重要作用。Meta分析显示新型5-HT4受体激动剂-普卢卡必利,有利于缓解慢性单纯性便秘,但对于STC的治疗证据仍显不足;非药物疗法,包括经皮电刺激、骶神经电刺激、粪菌移植等,操作复杂,临床难以普遍开展;手术治疗的复发率较高。而中医药治疗便秘有着悠久的历史,养阴润肠方是肛肠病专家朱秉宜教授治疗STC的经验方,临床疗效好。为进一步阐明其机制,本课题围绕养阴润肠方对ICC细胞和水通道蛋白3、9的影响展开研究,为更好地传承应用名医验方提供科学依据。目的:1.在动物水平,观察养阴润肠方对STC模型小鼠结肠组织SCF、c-kit表达量的影响、ICC超微结构的改变,以及水通道蛋白3和9(AQP3/9)表达量的影响;2.在细胞水平,观察养阴润肠方对于ICC体外增殖的影响,以及ICC胞内Ca2+浓度的变化。方法:1.通过复方地芬诺酯片建立STC小鼠模型,运用ELISA、免疫组化、qPCR、Western Blot观察小鼠结肠组织SCF、c-kit表达量的差异,运用透射电镜观察结肠组织中ICC超微结构的差异;2.运用免疫组化、qPCR、Westet Blot观察小鼠结肠组织AQP3和AQP9表达量的差异;3.采用酶解法获得ICC,免疫荧光鉴定,观察养阴润肠方对ICC增殖的影响;4.通过流式细胞分选获得纯化ICC,免疫荧光双染鉴定ICC,Ca2+荧光探针染色,激光共聚焦显微镜观察小鼠结肠ICC胞内Ca2+浓度。结果:1.通过复方地芬诺酯片成功建立STC模型小鼠,经养阴润肠方干预后,STC小鼠的粪便总量、粪便含水率增加(P<0.05),结肠传输速率加快(P<0.05);2.与模型组小鼠比较,养阴润肠方干预后,STC小鼠的血清SCF升高(P<0.05),结肠组织中SCF、c-kit的表达量均增加(P<0.05),并且损伤的ICC超微结构得到部分修复;3.与模型组小鼠比较,养阴润肠方干预后,STC小鼠结肠组织中AQP3的表达量明显下降(P<0.05),AQP9的表达量升高;4.单纯酶解法获得ICC,经养阴润肠方含药血清和直接给药的不同方式,均未能观察到养阴润肠方促进ICC的体外增殖;5.流式细胞分选获得纯化ICC,免疫荧光双染鉴定,经养阴润肠方水煎剂干预后,发现ICC胞内Ca2+浓度增加(P<0.01)。结论:1.养阴润肠方可以缓解STC小鼠的便秘症状,增加粪便总量和粪便含水量、促进肠动力的恢复;2.体外培养ICC,未观察到养阴润肠方直接促进细胞增殖,因此推测养阴润肠方在体内恢复结肠ICC细胞数量的机制可能是通过恢复SCF/c-kit通路而实现的,而并非直接作用于ICC;3.养阴润肠方可以增加ICC胞内Ca2+浓度,在一定程度上从功能学角度解释了养阴润肠方可能影响ICC的起搏功能;4.养阴润肠方可以降低STC小鼠结肠组织AQP3、升高AQP9的表达,影响肠道水液代谢,增加粪便含水量。更多还原

【Abstract】 The global incidence of chronic constipation is about 14%,of which slow transit constipation(STC)accounts for the first of four subtypes of chronic idiopathic constipation.Its main features are slow colonic transit,prolonged passage of stool,with or without abdominal distention,dry stool.Studies have shown that reduction of ICC cell number and abnormal expression of aquaporins in colon tissue play an important role in the pathology of STC.As a novel 5-HT4 receptor agonist,prucalopride is beneficial to the relief of chronic simple constipation,but the evidence for the treatment of STC is still insufficient.Non-drug therapy,including transcutaneous electrical stimulation,sacral nerve stimulation and fecal microbiota transplantation,which are difficult to carry out in clinical.The recurrence rate of surgical treatment of STC is higher.Chinese medicine has a long history of treating constipation.Yangyin Runchang Decoction is an anorectal specialist Zhu Bingyi’s experience in the treatment of STC,which is effective in clinical.This study aims at elucidating the mechanism of Yangyin Runchang Decoction on ICC and aquaporin 3/9 and at providing a scientific basis for better inheriting and application of the experienced prescription from famous experts.Objective:1.In animal,to observe the effects of Yangyin Runchang Decoction on the expression of SCF and c-kit in the colon tissue of mice with STC,the ultrastructural changes of ICC,and the expression of aquaporins 3 and 9(AQP3/9).2.At the cellular level,observe the effect of Yangyin Runchang Decoction on the proliferation of ICC in vitro,and the intracellular Ca2+ concentration of ICC.Methods:1.The STC mouse model was established by compound diphenoxylate tablets.The differences in SCF and c-kit expression in the colon tissue were observed by ELISA,immunohistochemistry,qPCR and Western Blot.The ultrastructure of ICC in colon tissue was observed by transmission electron microscope.2.Immunohistochemistry,qPCR,and Western Blot were used to observe the differences in the expression of AQP3 and AQP9 in mice colon tissues;3.ICC was obtained by enzymatic hydrolysis,and indentified by immunofluorescence.Observation the effect of Yangyin Runchang Decoction on ICC proliferation;4.The purified ICC was obtained by flow cytometric sorting.ICC was identified by immunofluorescence staining.Concentration of intracellular Ca2+ in the colonic ICC of mice was observed by laser confocal microscopy.Results:1.STC model mice were successfully established by compound diphenoxylate tablets.After intervention of Yangyin Runchang Decoction,the total feces and fecal water content of STC mice increased(P<0.05),and the colonic transmission rate increased(P<0.05);2.Compared with mice in the model group,serum SCF increased after intervention with Yangyin Runchang Decoction(P<0.05),the expression of SCF and c-kit in colon tissue were increased.(P<0.05),and the damaged ICC ultrastructure was partially repaired;3.Compared with mice in the model group,the expression of AQP3 in the colon tissue o was significantly decreased after intervention with Yangyin Runchang Decoction(P<0.05),and the expression of AQP9 was increased.4.In vitro proliferation of ICC was not observed with ICC obtained by enzymatic hydrolysis,no matter Yangyin Runchang Decoction was administrated by serum or directly.5.Purified ICC was obtained by flow cytometry and identified by immunofluorescence staining.After intervention of Yangying Runchang Decoction,the intracellular Ca2+concentration of ICC was increased(P<0.01).Conclusion:1.Yangyin Runchang Decoction can relieve the symptoms of constipation in STC mice,increase the total amount of feces and fecal water content,and promote the recovery of intestinal motility;2.No direct effect of Yangyin Runchang Decoction on cells was observed by In vitro culture of ICC.Therefore,it is inferred that the mechanism of Yangyin Runchang Decoction in restoring colonic ICC cell number in vivo may be achieved by restoring the SCF/c-kit pathway instead of directly affecting ICC;3.Yangyin Runchang Decoction can increase concentration of intracellular Ca2+ of ICC,to a certain extent,suggesting that Yangyin Runchang Decoction may affect the pacing function of ICC from a functional perspective;4.Yangyin Runchang Decoction can reduce AQP3 expression while increase AQP9 expression in colonic tissues of STC mice,which can increase fecal moisture content.更多还原