基于脑肠肽调节探讨功能性腹泻脾虚证的现代机理及中药干预作用

发布时间：2018-05-15 08:35

本文选题：功能性腹泻 + 脾虚证　；参考：《北京中医药大学》2017年硕士论文

【摘要】：功能性腹泻(Functional Diarrhea,FD)属于功能性肠病(Functional Bowel Disorders,FBDs)的一种,而功能性肠病是功能性胃肠病(Functional Gastrointestinal Disorders,FGIDs)表现在中段、下段消化道的肠道紊乱疾病。功能性腹泻是持续地或反复地出现排稀便(糊状便)或水样便,不伴有腹痛或腹部不适症状的综合征,诊断前症状出现至少6个月,近3个月症状符合以上标准。此病发病率较高,没有器质性病变,但产生的长期不适症状严重影响着患者的生活质量。其发病机制尚不明确,临床也缺少针对性的治疗药物。因此研究功能性腹泻的发病机制,寻找有效疗法成为研究热点。中医药干预治疗功能性腹泻有着良好的疗效,在基础研究方面也已成功建立功能性腹泻脾虚证病证结合动物模型,现代研究呈现出从整体水平至细胞水平乃至分子水平不断深入的发展趋势,故本研究在前期整体基础研究之上探索中药干预功能性腹泻脾虚证离体细胞水平的现代机理以及可能的疗效机制。理论研究研究一中医学对功能性腹泻的认识本研究通过从中医学的病名、病因、病机等方面对功能性腹泻进行更深入地认识,将功能性腹泻归于中医脾胃系病证"泄泻"一病,其病因多为感受外邪、饮食所伤、情志失调、禀赋不足、久病脏腑虚弱等,基本病机变化为脾病与湿盛,病位在肠,主病之脏属脾,主病之因为湿。功能性腹泻病程较长,久泄以脾虚为主,治法当以宜健脾。故将功能性腹泻脾虚证作为研究重点,选用参苓白术散加减的脾虚四号方进行治疗研究。研究二功能性腹泻与脾虚证的相关性研究本研究试图从诊断标准、临床症状、病因病机等方面来探讨功能性腹泻与脾虚证的相关性。功能性腹泻临床症状及较长病程与以大便溏泄为主的病程缠绵的脾虚证很是相似,脾虚证与功能性腹泻存在一致性。脾主运化是脾的主要生理功能之一,脾虚运化失司是导致脾虚证的主要原因。而脾虚失化会降低分解吸收精微物质的能力以及化湿能力,从而导致大便稀薄等功能性腹泻的症状,经过分析脾虚湿盛为功能性腹泻的核心病因病机,故本病当从脾论治,运用健脾化湿法。实验研究研究一功能性腹泻脾虚证病证结合动物模型的复制与评价目的成功复制并评价功能性腹泻脾虚证病证结合的动物模型。方法采用高乳糖饲料喂养加水环境小平台站立复合因素造模方法,观察大鼠一般状态、体重、摄食量、进水量,并测得腹泻指数,检测大鼠血清淀粉酶、D-木糖、乳酸含量作为判定功能性腹泻脾虚证动物模型的客观评价参考指标。结果模型组的大鼠出现淡黄色稀样便或水样便,毛发疏松不泽、神疲乏力、倦卧懒动、嗜睡、眯眼拱背、耳色淡暗等脾虚症状;较空白对照组,腹泻指数有非常显著性差异(P0.01);体重有非常显著性降低(P0.01),体重增长缓慢;摄食量较少;进水量较多;模型组大鼠血清淀粉酶含量降低有非常显著性差异(P0.01),D-木糖含量降低有显著性差异(P0.05),血清乳酸含量升高有非常显著性差异(P0.01)。结论已成功复制并评价功能性腹泻脾虚证病证结合的动物模型。研究二功能性腹泻脾虚证模型大鼠离体细胞的制备与原代培养目的成功提取并培养功能性腹泻脾虚证模型离体原代结肠平滑肌细胞以及下丘脑神经细胞。方法运用酶解法提取病证结合动物模型离体原代结肠玉平滑肌细胞以及下丘脑神经细胞。结果空白对照组、模型组结肠平滑肌细胞以及下丘脑神经细胞生长较稳定,状态尚可,可用于后期实验检测。结论已成功提取并培养功能性腹泻脾虚证模型离体原代结肠平滑肌细胞以及下丘脑神经细胞。研究三脾虚四号方含药血清干预功能性腹泻脾虚证模型大鼠离体细胞的作用机制研究目的探讨脾虚四号方含药血清干预功能性腹泻脾虚证模型离体原代结肠平滑肌细胞以及下丘脑神经细胞的治疗作用及可能的疗效机制。方法采用通法制备脾虚四号方含药血清,干预功能性腹泻脾虚证模型离体原代细胞。采用CCK-8法检测脾虚四号方含药血清干预模型离体原代结肠平滑肌细胞以及下丘脑神经细胞后,对其增殖活性的影响;采用ELISA、Real time PCR检测脾虚四号方含药血清干预模型离体原代结肠平滑肌细胞以及下丘脑神经细胞后,对其细胞上清中以及细胞中脑肠肽(CCK、VIP、SS、Ghrelin、SP、CGRP)的含量以及mRNA表达的影响。结果CCK-8结果显示:在结肠平滑肌细胞中,模型组较空白对照组OD值非常显著性降低(P0.01),脾虚四号方低、中、高剂量组以及蒙脱石散西药组较模型组OD值非常显著性升高(P0.01);在下丘脑神经细胞中,模型组较空白对照组OD值非常显著性降低(P0.01),脾虚四号方高剂量组较模型组OD值非常显著性升高(P0.01),其余含药血清组有升高趋势。ELISA结果显示:在结肠平滑肌细胞上清中,模型组CCK、VIP、SS、Ghrelin、SP的含量较空白对照组非常显著性降低(P0.01)CGRP的含量显著性降低(P0.05),脾虚四号方含药血清组较模型组其含量非常显著性升高或显著性升高(P0.01,P0.05);在下丘脑神经细胞上清中,模型组VIP、Ghrelin的含量较空白对照组非常显著性降低(P0.01)CCK的含量显著性降低(P0.05),模型组SS、SP、CGRP的含量较空白对照组非常显著性升高(P0.01),脾虚四号方含药血清组较模型组CCK、VIP、Ghrelin的含量非常显著性升高(P0.01),SS、SP、CGRP的含量非常显著性降低或显著性降低(P0.01,P0.05)。Real time PCR结果显示:在结肠平滑肌细胞中,模型组CCK、VIP、Ghrelin、SP、CGRP的mRNA表达较空白对照组非常显著性降低(P0.01)SS的表达显著性降低(P0.05),脾虚四号方含药血清组较模型组CCK、VIP、Ghrelin、SP、CGRP的mRNA表达非常显著性升高或显著性升高(P0.01,P0.05)SS有表达升高趋势;在下丘脑神经细胞中,模型组CCK、VIP的mRNA表达较空白对照组显著性降低(P0.05)Ghrelin有降低趋势,模型组SS、CGRP的mRNA表达较空白对照组非常显著性升高或显著性升高(P0.01,P0.05)SP有升高趋势,脾虚四号方含药血清组较模型组CCK的mRNA表达显著性升高(P0.05)VIP、Ghrelin有升高趋势,SS、CGRP的mRNA表达非常显著性降低或显著性降低(P0.01,P0.05)SP有降低趋势。结论模型结肠平滑肌细胞以及下丘脑神经细胞增殖活性较空白对照组显著性降低,而脾虚四号方含药血清能促进模型细胞增殖活性。模型结肠平滑肌细胞以及下丘脑神经细胞脑肠肽的含量及mRNA表达较空白对照组出现异常,而脾虚四号方含药血清能调节影响其异常的含量及mRNA表达。小结综合以上研究结果得出,中医学将功能性腹泻归于脾胃系病证"泄泻"一病,其主要病因病机为脾病与湿盛。功能性腹泻与脾脏密切相关,脾脏病变病证以虚证为主,着重于运化的失常。功能性腹泻与以大便溏泄为主的脾虚证具有一致性,脾虚湿盛为功能性腹泻的核心病因病机。通过成功复制并评价功能性腹泻脾虚证病证结合动物模型,建立起较稳定的病证结合动物模型体外原代结肠平滑肌细胞以及下丘脑神经细胞的分离培养方法。通过检测得知脾虚四号方含药血清能有效促进模型细胞增殖活性,提示其对模型离体原代结肠平滑肌细胞以及下丘脑神经细胞有一定治疗保护作用。脾虚四号方含药血清能影响模型原代细胞脑肠肽含量及mRNA表达,其可能通过升高或降低结肠平滑肌细胞以及下丘脑神经细胞的脑肠肽含量及mRNA表达来改善功能性腹泻脾虚证症状,调节脑肠肽可能是脾虚四号方疗效机制的作用靶点。脑肠肽紊乱与功能性腹泻脾虚证密切相关,脑肠肽失衡可能是脾虚失化脾虚证的现代机理。进行细胞水平的研究不仅拓宽了功能性腹泻脾虚证的现代研究还丰富了脾虚四号方中药干预治疗的发展。
[Abstract]:Functional diarrhea (Functional Diarrhea, FD) is one of the functional enteropathy (Functional Bowel Disorders, FBDs), while functional enteropathy is the functional gastrointestinal disease (Functional Gastrointestinal Disorders, FGIDs) in the middle, the lower digestive tract of the intestinal disorder. Functional diarrhea is persistent or recurring (paste). Symptoms of abdominal pain or abdominal discomfort were not accompanied by abdominal pain or abdominal discomfort. The symptoms before the diagnosis were at least 6 months, and the symptoms were in accordance with the above criteria for nearly 3 months. The incidence of this disease was high and no organic lesions, but the long-term discomfort symptoms seriously affected the quality of life of the patients. The pathogenesis of the disease was not clear and clinical was also lack of aim. Therefore, the study of the pathogenesis of functional diarrhea and the search for effective therapy have become a hot spot of research. Traditional Chinese medicine has a good effect on functional diarrhea in the treatment of functional diarrhea. In the basic research, it has also successfully established the model of the combination of functional diarrhea and spleen deficiency syndrome, and the present generation of research shows the level from the whole to the cell level. To the continuous development trend of molecular level, this study explored the modern mechanism and possible therapeutic mechanism of traditional Chinese medicine to interfere with the level of spleen deficiency syndrome in functional diarrhea. Functional diarrhoea is more deeply recognized, and functional diarrhea is attributed to the disease of "diarrhea" of the spleen and stomach syndrome of traditional Chinese medicine. The cause of the disease is mostly external evil, diet injury, emotional disorder, lack of endowment and weak viscera, basic pathogenesis changes to spleen and damp, the disease is in the intestines, the main disease is the spleen, the main disease is wet because wet. The course of diarrhoea is longer, and the spleen deficiency is the main part of the treatment. Therefore, the spleen deficiency syndrome of functional diarrhea is regarded as the focus of the study, and the spleen asthenia four is selected with the addition and subtraction of Shen Ling Baizhu powder. The study of the correlation between the two functional diarrhea and spleen deficiency syndrome is to try to get from the diagnostic criteria, clinical symptoms, etiological pathogenesis, and so on. To explore the correlation between functional diarrhea and spleen deficiency syndrome. The clinical symptoms and long course of functional diarrhoea are similar to that of the spleen deficiency syndrome with the course of loose stools. Spleen deficiency syndrome is consistent with functional diarrhea. Splenectomy is one of the main physiological functions of spleen. Spleen deficiency is the main cause of spleen deficiency, and spleen deficiency is the main cause of spleen deficiency. The deficiency can reduce the ability of decomposing and absorbing the subtle substances and the ability of humidification, which leads to the symptoms of functional diarrhea such as thin stool. After analyzing the core etiology of functional diarrhea, the spleen deficiency is considered as the core cause of diarrhea. Therefore, this disease should be treated from the spleen and use the wet method of strengthening spleen. The experimental study of a functional diarrhea spleen deficiency syndrome combined with animal models. Objective to replicate and evaluate the animal model of the combination of functional diarrhea and spleen deficiency syndrome. Methods using high lactose feed to feed the water environment small platform standing compound factor modeling method, the general state, weight, intake of food, water intake, and diarrhea index were observed in rats, and serum amylase, D- xylose and milk were detected in rats. The acid content was used as an objective evaluation index for evaluating the animal model of functional diarrhea spleen deficiency syndrome. The rats in the model group showed mild yellow dilute urine or water sample, loose hair, fatigue, lethargy, sleepiness, squinting arch back, light dark and other spleen deficiency symptoms, and the diarrhea index had a very significant difference compared with the blank control group (P0.0 1): the weight has a very significant reduction (P0.01), slow weight growth, less feeding and more water intake; the decrease of serum amylase content in model rats has a very significant difference (P0.01), the decrease of D- xylose content has significant difference (P0.05), the serum lactic acid content is very significant difference (P0.01). Conclusion the conclusion has been successfully replicated and evaluated. Animal model of combination of functional diarrhea and spleen deficiency syndrome. Study on the preparation and primary culture of the isolated cells of two functional diarrhea spleen deficiency rats and the aim to extract and cultivate the functional diarrhea spleen deficiency model of the isolated primary colonic smooth muscle cells and the hypothalamus nerve cells in vitro. The primary colonic smooth muscle cells and the hypothalamic nerve cells in the original colonic form were found in the blank control group. The growth of the colonic smooth muscle cells and the hypothalamus neurons in the model group was stable, and the state of the hypothalamus could be used for the later test. Conclusion the isolated primary colonic smooth muscle cells were successfully extracted and cultured in vitro from the model of functional diarrhea spleen deficiency. And the hypothalamic nerve cells. Study the effect mechanism of three spleen deficiency prescription four serum interfered on the rat model of functional diarrhea spleen deficiency model rats. Objective to explore the therapeutic effect and possible effect of spleen deficiency syndrome four prescription serum interfering on functional diarrhea of spleen deficiency syndrome model of isolated primary colonic smooth muscle cells and hypothalamus neurons in vitro Methods the effect mechanism was prepared by means of the prescription of spleen deficiency prescription four serum, interfering with functional diarrhea of spleen deficiency syndrome model in vitro primary cells. The effect of serum intervention model of spleen asthenia No. four on the proliferation of primary colonic smooth muscle cells and hypothalamus neurons in vitro was detected by CCK-8 method; ELISA, Real time PCR was used to detect the proliferation activity. The effect on the content of CCK, VIP, SS, Ghrelin, SP, CGRP and mRNA expression in the cell supernatant and the cells in the cell supernatant and the cells in the isolated primary colonic smooth muscle cells and the hypothalamic neurons. Results CCK-8 results showed that in the colonic smooth muscle cells, the model group was compared with the blank control group in the colon smooth muscle cells. Very significant reduction (P0.01), spleen asthenia No. four was low, middle, high dose group and Montmorillonite powder western medicine group were significantly higher than model group (P0.01). In the hypothalamic nerve cells, the model group was significantly lower than the blank control group (P0.01), and the high dose group of spleen deficiency No. four was significantly higher than the model group (P0.01). The results of the increase of the other serum containing.ELISA showed that in the supernatant of colonic smooth muscle cells, the content of CCK, VIP, SS, Ghrelin, SP in the model group was significantly lower than that in the blank control group (P0.01), the content of CGRP was significantly decreased (P0.05), and the content of serum containing spleen deficiency four serum group was significantly higher or significantly higher than that of the model group. High (P0.01, P0.05); in the hypothalamus nerve cell supernatant, the content of VIP, Ghrelin in the model group was significantly lower than that in the blank control group (P0.01), the content of CCK was significantly decreased (P0.05). The content of SS, SP, CGRP in the model group was significantly higher than that in the blank control group (P0.01), and the serum group containing the spleen deficiency No. four was more than the model group. The content of SS, SP, and CGRP decreased significantly or significantly decreased (P0.01, P0.05).Real time PCR results showed that in the colonic smooth muscle cells, the expressions of CCK, VIP, Ghrelin, and CCK were significantly lower than those in the blank control group. The expression of.Real time was significantly lower than that of the blank control group, and the spleen deficiency was four square. Compared with model group CCK, VIP, Ghrelin, SP, CGRP, mRNA expression increased significantly or significantly increased (P0.01, P0.05) SS expressed in the model group, and in the hypothalamic nerve cells, the expression of CCK and VIP mRNA in the model group decreased significantly than that in the blank control group. The control group was significantly elevated or significantly elevated (P0.01, P0.05) SP increased, the mRNA expression in the spleen deficiency group four serum group was significantly higher than that of the model group CCK (P0.05) VIP, Ghrelin had a tendency to rise, SS, CGRP mRNA expression decreased significantly or significantly decreased. Conclusion the model colon is flat. The proliferation activity of the smooth muscle cells and the hypothalamus nerve cells was significantly lower than that in the blank control group, while the serum containing the spleen deficiency four prescription could promote the proliferation activity of the model cells. The content of the model colon smooth muscle cells and the hypothalamus nerve cells were abnormal in the mRNA expression compared with the blank control group, while the serum level of the spleen asthenia No. four prescription contained serum. The results showed that the main causes of functional diarrhea in the spleen and stomach disease syndrome "diarrhea" disease, the main cause of the pathogenesis of spleen and dampness. Functional diarrhea is closely related to the spleen and the spleen, the spleen disease syndrome is mainly in virtual syndrome, especially in the dysfunctional disorder. Functional diarrhea and mRNA The spleen deficiency syndrome with loose stools is consistent and the spleen deficiency is the core pathogenesis of functional diarrhea. Through the successful replication and evaluation of functional diarrhea spleen deficiency syndrome combined with animal models, a more stable disease syndrome combined animal model was established for the isolation and culture of the original colonic smooth muscle cells and the hypothalamus nerve cells in vitro. Methods. It was found that the serum of prescription four of spleen deficiency can effectively promote the proliferation activity of the model cells, suggesting that it has certain protective effects on the model isolated primary colonic smooth muscle cells and the hypothalamus nerve cells. The serum of the spleen deficiency prescription No. four can affect the content of the brain gut peptide and the expression of mRNA in the primary cells of the model. The content of brain intestinal peptide and mRNA expression in the colonic smooth muscle cells and hypothalamus nerve cells can improve the symptoms of spleen deficiency syndrome of functional diarrhea. The regulation of brain intestinal peptide may be the target of the therapeutic mechanism of spleen deficiency four prescription. The disorder of brain gut peptide is closely related to the spleen deficiency syndrome of functional diarrhea, and the imbalance of the brain gut peptide may be the spleen deficiency and the spleen deficiency syndrome. The study of cell level not only widens the modern research on the syndrome of spleen deficiency of functional diarrhea, but also enriches the development of the intervention treatment of Chinese traditional Chinese medicine No. four of spleen deficiency.

【学位授予单位】：北京中医药大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R259

【参考文献】