本文選題：功能性腹瀉 + 脾虛證��； 參考：《北京中醫(yī)藥大學(xué)》2017年碩士論文

【摘要】：功能性腹瀉(Functional Diarrhea,FD)屬于功能性腸病(Functional Bowel Disorders,FBDs)的一種,而功能性腸病是功能性胃腸病(Functional Gastrointestinal Disorders,FGIDs)表現(xiàn)在中段、下段消化道的腸道紊亂疾病。功能性腹瀉是持續(xù)地或反復(fù)地出現(xiàn)排稀便(糊狀便)或水樣便,不伴有腹痛或腹部不適癥狀的綜合征,診斷前癥狀出現(xiàn)至少6個(gè)月,近3個(gè)月癥狀符合以上標(biāo)準(zhǔn)。此病發(fā)病率較高,沒有器質(zhì)性病變,但產(chǎn)生的長期不適癥狀嚴(yán)重影響著患者的生活質(zhì)量。其發(fā)病機(jī)制尚不明確,臨床也缺少針對性的治療藥物。因此研究功能性腹瀉的發(fā)病機(jī)制,尋找有效療法成為研究熱點(diǎn)。中醫(yī)藥干預(yù)治療功能性腹瀉有著良好的療效,在基礎(chǔ)研究方面也已成功建立功能性腹瀉脾虛證病證結(jié)合動(dòng)物模型,現(xiàn)代研究呈現(xiàn)出從整體水平至細(xì)胞水平乃至分子水平不斷深入的發(fā)展趨勢,故本研究在前期整體基礎(chǔ)研究之上探索中藥干預(yù)功能性腹瀉脾虛證離體細(xì)胞水平的現(xiàn)代機(jī)理以及可能的療效機(jī)制。理論研究研究一中醫(yī)學(xué)對功能性腹瀉的認(rèn)識本研究通過從中醫(yī)學(xué)的病名、病因、病機(jī)等方面對功能性腹瀉進(jìn)行更深入地認(rèn)識,將功能性腹瀉歸于中醫(yī)脾胃系病證"泄瀉"一病,其病因多為感受外邪、飲食所傷、情志失調(diào)、稟賦不足、久病臟腑虛弱等,基本病機(jī)變化為脾病與濕盛,病位在腸,主病之臟屬脾,主病之因?yàn)闈�。功能性腹瀉病程較長,久泄以脾虛為主,治法當(dāng)以宜健脾。故將功能性腹瀉脾虛證作為研究重點(diǎn),選用參苓白術(shù)散加減的脾虛四號方進(jìn)行治療研究。研究二功能性腹瀉與脾虛證的相關(guān)性研究本研究試圖從診斷標(biāo)準(zhǔn)、臨床癥狀、病因病機(jī)等方面來探討功能性腹瀉與脾虛證的相關(guān)性。功能性腹瀉臨床癥狀及較長病程與以大便溏泄為主的病程纏綿的脾虛證很是相似,脾虛證與功能性腹瀉存在一致性。脾主運(yùn)化是脾的主要生理功能之一,脾虛運(yùn)化失司是導(dǎo)致脾虛證的主要原因。而脾虛失化會(huì)降低分解吸收精微物質(zhì)的能力以及化濕能力,從而導(dǎo)致大便稀薄等功能性腹瀉的癥狀,經(jīng)過分析脾虛濕盛為功能性腹瀉的核心病因病機(jī),故本病當(dāng)從脾論治,運(yùn)用健脾化濕法。實(shí)驗(yàn)研究研究一功能性腹瀉脾虛證病證結(jié)合動(dòng)物模型的復(fù)制與評價(jià)目的成功復(fù)制并評價(jià)功能性腹瀉脾虛證病證結(jié)合的動(dòng)物模型。方法采用高乳糖飼料喂養(yǎng)加水環(huán)境小平臺(tái)站立復(fù)合因素造模方法,觀察大鼠一般狀態(tài)、體重、攝食量、進(jìn)水量,并測得腹瀉指數(shù),檢測大鼠血清淀粉酶、D-木糖、乳酸含量作為判定功能性腹瀉脾虛證動(dòng)物模型的客觀評價(jià)參考指標(biāo)。結(jié)果模型組的大鼠出現(xiàn)淡黃色稀樣便或水樣便,毛發(fā)疏松不澤、神疲乏力、倦臥懶動(dòng)、嗜睡、瞇眼拱背、耳色淡暗等脾虛癥狀;較空白對照組,腹瀉指數(shù)有非常顯著性差異(P0.01);體重有非常顯著性降低(P0.01),體重增長緩慢;攝食量較少;進(jìn)水量較多;模型組大鼠血清淀粉酶含量降低有非常顯著性差異(P0.01),D-木糖含量降低有顯著性差異(P0.05),血清乳酸含量升高有非常顯著性差異(P0.01)。結(jié)論已成功復(fù)制并評價(jià)功能性腹瀉脾虛證病證結(jié)合的動(dòng)物模型。研究二功能性腹瀉脾虛證模型大鼠離體細(xì)胞的制備與原代培養(yǎng)目的成功提取并培養(yǎng)功能性腹瀉脾虛證模型離體原代結(jié)腸平滑肌細(xì)胞以及下丘腦神經(jīng)細(xì)胞。方法運(yùn)用酶解法提取病證結(jié)合動(dòng)物模型離體原代結(jié)腸玉平滑肌細(xì)胞以及下丘腦神經(jīng)細(xì)胞。結(jié)果空白對照組、模型組結(jié)腸平滑肌細(xì)胞以及下丘腦神經(jīng)細(xì)胞生長較穩(wěn)定,狀態(tài)尚可,可用于后期實(shí)驗(yàn)檢測。結(jié)論已成功提取并培養(yǎng)功能性腹瀉脾虛證模型離體原代結(jié)腸平滑肌細(xì)胞以及下丘腦神經(jīng)細(xì)胞。研究三脾虛四號方含藥血清干預(yù)功能性腹瀉脾虛證模型大鼠離體細(xì)胞的作用機(jī)制研究目的探討脾虛四號方含藥血清干預(yù)功能性腹瀉脾虛證模型離體原代結(jié)腸平滑肌細(xì)胞以及下丘腦神經(jīng)細(xì)胞的治療作用及可能的療效機(jī)制。方法采用通法制備脾虛四號方含藥血清,干預(yù)功能性腹瀉脾虛證模型離體原代細(xì)胞。采用CCK-8法檢測脾虛四號方含藥血清干預(yù)模型離體原代結(jié)腸平滑肌細(xì)胞以及下丘腦神經(jīng)細(xì)胞后,對其增殖活性的影響;采用ELISA、Real time PCR檢測脾虛四號方含藥血清干預(yù)模型離體原代結(jié)腸平滑肌細(xì)胞以及下丘腦神經(jīng)細(xì)胞后,對其細(xì)胞上清中以及細(xì)胞中腦腸肽(CCK、VIP、SS、Ghrelin、SP、CGRP)的含量以及mRNA表達(dá)的影響。結(jié)果CCK-8結(jié)果顯示:在結(jié)腸平滑肌細(xì)胞中,模型組較空白對照組OD值非常顯著性降低(P0.01),脾虛四號方低、中、高劑量組以及蒙脫石散西藥組較模型組OD值非常顯著性升高(P0.01);在下丘腦神經(jīng)細(xì)胞中,模型組較空白對照組OD值非常顯著性降低(P0.01),脾虛四號方高劑量組較模型組OD值非常顯著性升高(P0.01),其余含藥血清組有升高趨勢。ELISA結(jié)果顯示:在結(jié)腸平滑肌細(xì)胞上清中,模型組CCK、VIP、SS、Ghrelin、SP的含量較空白對照組非常顯著性降低(P0.01)CGRP的含量顯著性降低(P0.05),脾虛四號方含藥血清組較模型組其含量非常顯著性升高或顯著性升高(P0.01,P0.05);在下丘腦神經(jīng)細(xì)胞上清中,模型組VIP、Ghrelin的含量較空白對照組非常顯著性降低(P0.01)CCK的含量顯著性降低(P0.05),模型組SS、SP、CGRP的含量較空白對照組非常顯著性升高(P0.01),脾虛四號方含藥血清組較模型組CCK、VIP、Ghrelin的含量非常顯著性升高(P0.01),SS、SP、CGRP的含量非常顯著性降低或顯著性降低(P0.01,P0.05)。Real time PCR結(jié)果顯示:在結(jié)腸平滑肌細(xì)胞中,模型組CCK、VIP、Ghrelin、SP、CGRP的mRNA表達(dá)較空白對照組非常顯著性降低(P0.01)SS的表達(dá)顯著性降低(P0.05),脾虛四號方含藥血清組較模型組CCK、VIP、Ghrelin、SP、CGRP的mRNA表達(dá)非常顯著性升高或顯著性升高(P0.01,P0.05)SS有表達(dá)升高趨勢;在下丘腦神經(jīng)細(xì)胞中,模型組CCK、VIP的mRNA表達(dá)較空白對照組顯著性降低(P0.05)Ghrelin有降低趨勢,模型組SS、CGRP的mRNA表達(dá)較空白對照組非常顯著性升高或顯著性升高(P0.01,P0.05)SP有升高趨勢,脾虛四號方含藥血清組較模型組CCK的mRNA表達(dá)顯著性升高(P0.05)VIP、Ghrelin有升高趨勢,SS、CGRP的mRNA表達(dá)非常顯著性降低或顯著性降低(P0.01,P0.05)SP有降低趨勢。結(jié)論模型結(jié)腸平滑肌細(xì)胞以及下丘腦神經(jīng)細(xì)胞增殖活性較空白對照組顯著性降低,而脾虛四號方含藥血清能促進(jìn)模型細(xì)胞增殖活性。模型結(jié)腸平滑肌細(xì)胞以及下丘腦神經(jīng)細(xì)胞腦腸肽的含量及mRNA表達(dá)較空白對照組出現(xiàn)異常,而脾虛四號方含藥血清能調(diào)節(jié)影響其異常的含量及mRNA表達(dá)。小結(jié)綜合以上研究結(jié)果得出,中醫(yī)學(xué)將功能性腹瀉歸于脾胃系病證"泄瀉"一病,其主要病因病機(jī)為脾病與濕盛。功能性腹瀉與脾臟密切相關(guān),脾臟病變病證以虛證為主,著重于運(yùn)化的失常。功能性腹瀉與以大便溏泄為主的脾虛證具有一致性,脾虛濕盛為功能性腹瀉的核心病因病機(jī)。通過成功復(fù)制并評價(jià)功能性腹瀉脾虛證病證結(jié)合動(dòng)物模型,建立起較穩(wěn)定的病證結(jié)合動(dòng)物模型體外原代結(jié)腸平滑肌細(xì)胞以及下丘腦神經(jīng)細(xì)胞的分離培養(yǎng)方法。通過檢測得知脾虛四號方含藥血清能有效促進(jìn)模型細(xì)胞增殖活性,提示其對模型離體原代結(jié)腸平滑肌細(xì)胞以及下丘腦神經(jīng)細(xì)胞有一定治療保護(hù)作用。脾虛四號方含藥血清能影響模型原代細(xì)胞腦腸肽含量及mRNA表達(dá),其可能通過升高或降低結(jié)腸平滑肌細(xì)胞以及下丘腦神經(jīng)細(xì)胞的腦腸肽含量及mRNA表達(dá)來改善功能性腹瀉脾虛證癥狀,調(diào)節(jié)腦腸肽可能是脾虛四號方療效機(jī)制的作用靶點(diǎn)。腦腸肽紊亂與功能性腹瀉脾虛證密切相關(guān),腦腸肽失衡可能是脾虛失化脾虛證的現(xiàn)代機(jī)理。進(jìn)行細(xì)胞水平的研究不僅拓寬了功能性腹瀉脾虛證的現(xiàn)代研究還豐富了脾虛四號方中藥干預(yù)治療的發(fā)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.

【學(xué)位授予單位】：北京中醫(yī)藥大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R259

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