本文關(guān)鍵詞：針刺足三里穴治療功能性消化不良大鼠實驗研究　出處：《大連醫(yī)科大學(xué)》2016年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 功能性消化不良 針刺足三里穴 腸道敏感性 Cajal間質(zhì)細(xì)胞 免疫 差異基因 信號通路

【摘要】：背景:功能性消化不良(functional dyspepsia,FD)是臨床最常見的一種功能性胃腸病,是指具有上腹痛、上腹脹、早飽、噯氣、食欲不振、惡心、嘔吐等不適癥狀,經(jīng)檢查排除引起上述癥狀的器質(zhì)性疾病的一組臨床綜合征,與中醫(yī)的脾氣虛證類似。癥狀可持續(xù)或反復(fù)發(fā)作,嚴(yán)重影響患者的生活質(zhì)量,降低工作效率,增加了醫(yī)療資源的消耗。研究發(fā)現(xiàn),FD患者存在胃腸道的動力異常及內(nèi)臟高敏癥,但該病的確切發(fā)病機(jī)制目前仍不清楚。針刺作為中國傳統(tǒng)醫(yī)學(xué)的一部分,在我國應(yīng)用已有千余年的歷史。足三里穴定位為犢鼻下3寸,脛骨前嵴外1橫指處。中西醫(yī)結(jié)合臨床實踐中,發(fā)現(xiàn)針刺足陽明胃經(jīng)在功能性胃腸疾病方面具有自己獨特的優(yōu)勢,尤其是針刺足三里穴應(yīng)用較為廣泛。很多研究均顯示,針刺足三里穴可以有效緩解功能性胃腸病,對其確切作用機(jī)制,目前亦不完全清楚。目的:通過水浸法建立FD大鼠模型,觀察大鼠日均攝食量、體重、腸道敏感性、小腸Cajal間質(zhì)細(xì)胞、血清免疫指標(biāo)的變化,通過表達(dá)譜基因芯片檢測差異基因、差異基因的分子功能、差異基因參與的信號通路,進(jìn)一步研究FD的發(fā)病機(jī)制。對FD大鼠進(jìn)行針刺足三里穴治療,觀察大鼠日均攝食量、體重、腸道敏感性、小腸Cajal間質(zhì)細(xì)胞、血清免疫指標(biāo)的變化,通過表達(dá)譜基因芯片檢測差異基因、差異基因的分子功能、差異基因參與的信號通路,探討針刺足三里穴治療FD疾病的機(jī)制。方法:成年Wistar大鼠被分成對照(control)組、模型(model)組、針刺足三里穴(acupuncture,AP)組、非經(jīng)非穴針刺(sham acupuncture,sham-AP)組。除了control組,將其它組的大鼠通過水浸法建立FD模型。建立FD模型后,監(jiān)測control組與model組大鼠攝食量、體重,評估大鼠腸道敏感性。通過結(jié)直腸擴(kuò)張(colorectal distention,CRD)來誘發(fā)急性內(nèi)臟痛,用大鼠對急性內(nèi)臟痛的腹部回撤反射(abdominal withdrawal reflex,AWR)來評價腸道敏感性。取大鼠小腸組織,進(jìn)行免疫熒光染色,共聚焦顯微鏡檢測深肌層cajal間質(zhì)細(xì)胞形態(tài)及數(shù)量。檢測血清igg、igm、il-2和il-6水平。然后組間結(jié)果進(jìn)行比較。取大鼠小腸組織,進(jìn)行基因表達(dá)譜芯片檢測,篩選model組與control組的差異基因,查找其分子功能,篩選差異基因相關(guān)信號通路。建立fd模型后,ap組大鼠針刺足三里穴15天,每天一次,每次時間為20分鐘。sham-ap組大鼠于非經(jīng)非穴點針刺15天,對model組大鼠進(jìn)行束縛但不予針刺。對control組大鼠自然飼養(yǎng)。監(jiān)測大鼠攝食量、體重,評估大鼠腸道敏感性。取大鼠小腸組織,進(jìn)行免疫熒光染色,共聚焦顯微鏡檢測深肌層cajal間質(zhì)細(xì)胞形態(tài)及數(shù)量。檢測血清igg、igm、il-2和il-6水平。然后將control組、model組、ap組和sham-ap組間數(shù)據(jù)進(jìn)行比較。取大鼠小腸組織,進(jìn)行基因表達(dá)譜芯片檢測,篩選ap組與model組的差異基因,查找其分子功能,篩選差異基因相關(guān)信號通路。結(jié)果:1.model組大鼠的日均攝食量明顯低于control組(t=-5.360p0.01);體重明顯低于control組(t=-3.144p0.05);腸道敏感性明顯高于control組。在model組,當(dāng)crd的壓力為20、40、60或80mmhg時,awr評分均顯著高于control組(t=5.00,p0.01;t=2.71,p0.05;t=2.71,p0.05;t=3.16,p0.05)。再經(jīng)過針刺足三里穴15天后,ap組(針刺組)大鼠的日均攝食量明顯高于model組(非針刺組)(p0.05);體重明顯高于model組(p0.05);當(dāng)crd的壓力為20mmhg時,ap組大鼠的腸道高敏癥較model組無明顯差異(p0.05)。當(dāng)crd的壓力為40、60或80mmhg時,ap組大鼠的腸道高敏癥較model組減輕(p0.05,p0.05,p0.05)。2.model組大鼠的小腸cajal間質(zhì)細(xì)胞形態(tài)明顯不正常且數(shù)量低于control組。cajal間質(zhì)細(xì)胞的免疫熒光值顯著低于control組(t=-21.45,p0.01)。再經(jīng)過針刺足三里穴15天后,ap組大鼠的小腸cajal間質(zhì)細(xì)胞形態(tài)恢復(fù)且數(shù)量明顯高于model組。cajal間質(zhì)細(xì)胞的免疫熒光值顯著高于model組(p0.05)。3.model組大鼠的血清igg、igm和il-2水平明顯低于control組(t=-11.67,p0.01;t=-11.26,p0.01;t=-4.44,p0.01),血清il-6水平明顯高于control組(t=5.23,p0.01)。再經(jīng)過針刺足三里穴15天后,ap組大鼠的血清igg、igm和il-2水平明顯高于model組(p0.05;p0.05;p0.05),血清il-6水平明顯低于model組(p0.05)。4.小腸組織基因表達(dá)譜芯片檢測:排除無geneid的基因,以差異倍數(shù)(foldchange,fc)2或0.5,p0.01為標(biāo)準(zhǔn),model組大鼠和control組相比,篩選差異基因,有255個差異基因,其中上調(diào)基因11個,下調(diào)基因244個。上調(diào)最顯著的10個基因是rgd1559622、tsks、gramd2、colq、kcnq4、mecom、tulp1、thrb、acot1、lss,下調(diào)最顯著的10基因是loc100362150、mcpt2、igh-6、mcpt1l4、cpa3、mcpt8l2、fcer1a、loc363827、gpr174、loc100363695。篩選出差異基因可能相關(guān)的信號通路19條,為(1)renin-angiotensinsystem(2)primaryimmunodeficiency(3)staphylococcusaureusinfection(4)intestinalimmunenetworkforigaproduction-rattusnorvegicus(rat)(5)naturalkillercellmediatedcytotoxicity(6)nod-likereceptorsignalingpathway-rattusnorvegicus(rat)(7)celladhesionmolecules(cams)-rattusnorvegicus(rat)(8)etherlipidmetabolism(9)fcepsilonrisignalingpathway(10)leukocytetransendothelialmigration(11)cytokine-cytokinereceptorinteraction-rattusnorvegicus(rat)(12)toll-likereceptorsignalingpathway(13)tcellreceptorsignalingpathway(14)chemokinesignalingpathway(15)phagosome(16)fcgammar-mediatedphagocytosis-rattusnorvegicus(rat)(17)antigenprocessingandpresentation(18)lysosome-rattusnorvegicus(rat)(19)regulationofactincytoskeleton。共15條信號通路明顯與免疫反應(yīng)相關(guān),其中3條信號通路與肥大細(xì)胞反應(yīng)相關(guān);共5條信號通路與細(xì)胞增殖、分化、凋亡相關(guān);共2條信號通路與細(xì)胞內(nèi)活動、細(xì)胞內(nèi)代謝、細(xì)胞運動有關(guān)。ap組大鼠和model組相比,篩選差異基因,有169個差異基因,其中上調(diào)基因96個,下調(diào)基因73個。上調(diào)最顯著的10個基因是slc34a2、trpv6、gucy2g、hbb、hbb-b1、loc287167、rgd1564463、akp3、alas2、gsg1,下調(diào)最顯著的10個基因是acot1、loc690226、hmgcs2、gbx1、retsat、klrb1b、ppp1r1c、slc27a2、loc683474、masp2。篩選出差異基因可能相關(guān)的信號通路25條,為(1)biosynthesisofunsaturatedfattyacids(2)steroidbiosynthesis(3)synthesisanddegradationofketonebodies(4)glycosphingolipidbiosynthesis-globo series(5)PPAR signaling pathway(6)Fatty acid metabolism(7)Butanoate metabolism(8)Mineral absorption(9)Valine,leucine and isoleucine degradation(10)Steroid hormone biosynthesis(11)Ascorbate and aldarate metabolism(12)Porphyrin and chlorophyll metabolism(13)Glycosphingolipid biosynthesis-lacto and neolacto series(14)Retinol metabolism(15)ABC transporters(16)Pentose and glucuronate interconversions(17)Drug metabolismcytochrome P450-Rattus norvegicus(rat)(18)Drug metabolism-other enzymes-Rattus norvegicus(rat)(19)Metabolism of xenobiotics by cytochrome P450-Rattus norvegicus(rat)(20)Carbohydrate digestion and absorption(21)Peroxisome-Rattus norvegicus(rat)(22)Fat digestion and absorption(23)Starch and sucrose metabolism(24)Arachidonic acid metabolism(25)Glycerophospholipid metabolism。共8條信號通路與脂類代謝有關(guān);共7條信號通路與碳水化合物、多糖代謝有關(guān);共3條信號通路與激素、酶等代謝有關(guān);共2條信號通路與細(xì)胞內(nèi)轉(zhuǎn)運、分解代謝,細(xì)胞膜活動等相關(guān);1條信號通路與細(xì)胞增殖、分化等活動相關(guān)。結(jié)論:針刺足三里穴能夠提高FD大鼠攝食量和體重,改善FD大鼠腸道高敏癥,是治療FD的理想治療方式;在FD大鼠,小腸Cajal間質(zhì)細(xì)胞形態(tài)異常且數(shù)量明顯降低,這可能是FD大鼠胃腸運動功能障礙的原因。在FD大鼠,存在免疫功能低下及免疫功能失調(diào),這可能是FD內(nèi)臟高敏癥的原因;針刺足三里穴顯示出對FD較好的治療效果,提示治療的機(jī)制可能與針刺足三里穴能夠改善FD大鼠胃腸道Cajal間質(zhì)細(xì)胞損傷和改善FD大鼠免疫功能有關(guān);FD疾病的分子機(jī)制方面,所篩選信號通路與免疫反應(yīng),肥大細(xì)胞反應(yīng),細(xì)胞增殖分化等方面相關(guān),可結(jié)合相關(guān)差異基因及具體信號通路,進(jìn)一步深化研究。針刺足三里穴治療FD疾病的分子機(jī)制方面,所篩選信號通路與營養(yǎng)代謝調(diào)節(jié)、吸收,激素分泌調(diào)節(jié)等相關(guān),可結(jié)合相關(guān)的差異基因及具體信號通路,進(jìn)一步深化研究。
[Abstract]:Background: functional dyspepsia (functional dyspepsia FD) is one of the most common functional gastrointestinal disorders, are abdominal pain, abdominal bloating, early satiety, belching, loss of appetite, nausea, vomiting and other symptoms, after examination to exclude organic diseases caused by the symptoms of a group of clinical comprehensive similar to the traditional Chinese Medicine syndrome, spleen qi deficiency symptoms. Sustainable or recurrent, seriously affecting the quality of life of patients, reduce work efficiency, increase the consumption of medical resources. The study found that dynamic FD patients have gastrointestinal abnormalities and visceral hypersensitivity disease, but the exact pathogenesis of the disease remains unclear. As acupuncture a part of Chinese traditional medicine, has been applied in China for thousands of years of history. Zusanli positioning for the calf nose 3 inch, anterior tibial crest 1 finger. Combining traditional Chinese and Western medicine clinical practice, found that acupuncture of Foot Yangming Meridian in functional gastrointestinal It has its own unique advantages of diseases, especially acupuncture is widely used. Many studies have shown that acupuncture can effectively alleviate the functional gastrointestinal disease, the exact mechanism of action is also unclear. Objective: the rat model of FD was established by water immersion, rats were observed daily food intake. Body weight, intestinal sensitivity, interstitial cells of Cajal, changes of serum immune indexes, the gene expression spectrum of gene chip detection differences, molecular function of differential genes, signal transduction genes were involved in the pathogenesis of FD, further research. The acupuncture treatment of FD rats, rats were observed daily food intake. Body weight, intestinal sensitivity, interstitial cells of Cajal, changes of serum immune indexes, the gene expression spectrum of gene chip detection differences, molecular function of differential genes, genes involved in the signal difference To explore the mechanism of acupuncture treatment, FD disease Zusanli. Methods: adult Wistar rats were divided into control group (control), model group (model), acupuncture group (acupuncture, AP), non acupoint acupuncture group (sham acupuncture, sham-AP). In group control, the other big group through the establishment of rat FD model of water immersion method. After the establishment of FD model, monitoring of control group and model group rats food intake, body weight, assessment of bowel sensitivity in rats by colorectal distension (colorectal distention CRD) to induce acute visceral pain in rats, on acute visceral pain Abdominal withdrawal reflex (Abdominal withdrawal reflex AWR), to evaluate the intestinal sensitivity. Small intestine of rats by immunofluorescence staining and confocal microscopy to detect deep myometrial Cajal interstitial cell morphology and quantity. The detection of serum IgG, IgM, IL-2 and IL-6 level. Then the results were compared between groups. The rat small intestine Tissue microarray detection of genes differentially expressed genes of model group and control group, the molecular function of search, screening differential gene related signal pathway. After the establishment of FD model, AP rats by acupuncture for 15 days, once a day, 20 minutes for each time the rats in the.Sham-ap group non acupoint acupuncture point for 15 days, the rats in group model were bound but without acupuncture. The rats of control group rats were monitored for natural feeding. Food intake, body weight, assessment of bowel sensitivity in rats. The intestinal tissue of rats, immunofluorescence staining, confocal microscopy, deep myometrial Cajal interstitial cell morphology and quantity. Detection of serum IgG, IgM, IL-2 and IL-6. Then the control group, model group, AP group and sham-ap group were compared between the data. The intestinal tissue of rats, for microarray detection of gene differentially expressed genes, AP and model groups, find the molecular function, Screening of differential genes related to signal pathway. Results: the average daily intake of rats in 1.model group were significantly lower than that in group control (t=-5.360p0.01); body weight was significantly lower than control group (t=-3.144p0.05); intestinal sensitivity was significantly higher than that of control group. In model group, when CRD pressure is 20,40,60 or 80mmHg, AWR scores were significantly higher than that of group control (t=5.00 t=2.71, P0.05, P0.01; t=2.71, P0.05; t=3.16, P0.05;). After acupuncture in 15 days, AP group (acupuncture group) average daily food intake in rats was significantly higher than that of model group (non acupuncture group) (P0.05); body weight was significantly higher than that of model group (P0.05); when the pressure is CRD 20mmhg, compared with model group, AP group have no significant differences in rat intestinal Gao Min disease (P0.05). When CRD pressure is 40,60 or 80mmHg, AP rats intestinal Gao Min disease compared with the model group (P0.05, P0.05, P0.05).2.model rats intestinal interstitial cells of Cajal form is obviously not normal and The number is lower than the control group.Cajal immunofluorescence of Leydig cells was significantly lower than that of group control (t=-21.45, P0.01). After acupuncture 15 days later, the rats in the AP group of interstitial cells of Cajal morphology and the number of recovery was significantly higher than model group.Cajal immunofluorescence of Leydig cells was significantly higher than group model (P0.05) in serum IgG rats of group.3.model, IgM and IL-2 levels were significantly lower than that of group control (t=-11.67, P0.01; t=-11.26, P0.01; t=-4.44, P0.01), the serum level of IL-6 was significantly higher than that of group control (t=5.23, P0.01). After 15 days after acupuncture, serum IgG of rats in the AP group, IgM and IL-2 were significantly higher than that of group model (P0.05; P0.05; P0.05), the serum level of IL-6 was significantly lower than that of group model (P0.05).4. gene expression microarray detection of intestinal tissue: the exclusion of non geneid genes with fold difference (foldchange, FC) 2 or 0.5, P0.01 as the standard, the rats in group model and group control Compared with the screening of differentially expressed genes, 255 differentially expressed genes, including 11 up-regulated genes and 244 down regulated genes. 10 genes were significantly up-regulated rgd1559622, TSKS, gramd2, COLQ, KCNQ4, MECom, tulp1, thrb, acot1, LSS, down 10 gene was loc100362150, mcpt2, igh-6. Mcpt1l4, cpa3, mcpt8l2, FCER1A, loc363827, gpr174, loc100363695. genes were screened the possible signal pathway related 19, as (1) renin-angiotensinsystem (2) primaryimmunodeficiency (3) staphylococcusaureusinfection (4) intestinalimmunenetworkforigaproduction-rattusnorvegicus (rat) (5) naturalkillercellmediatedcytotoxicity (6) nod-likereceptorsignalingpathway-rattusnorvegicus (rat) (7) celladhesionmolecules (CAMs) -rattusnorvegicus (rat) (8) etherlipidmetabolism (9) fcepsilonrisignalingpathway (10) leukocytetransendothelialmigration (11) cyto Kine-cytokinereceptorinteraction-rattusnorvegicus (rat) (12) toll-likereceptorsignalingpathway (13) tcellreceptorsignalingpathway (14) chemokinesignalingpathway (15) phagosome (16) fcgammar-mediatedphagocytosis-rattusnorvegicus (rat) (17) antigenprocessingandpresentation (18) lysosome-rattusnorvegicus (rat) (19) regulationofactincytoskeleton. 15 signaling pathway and the immune response was related to the 3 signal pathways and mast cell reaction; a total of 5 apoptosis signaling pathway and cell proliferation, differentiation, related; a total of 2 signaling pathways and cell activity, cellular metabolism, cell motility related compared to rats in group.Ap and group model, screening of differentially expressed genes, 169 differentially expressed genes, including 96 up-regulated genes and 73 down regulated genes and 10 genes. The most significant increase is SLC34A2, TRPV6, gucy2g, HBB, hbb-b1, loc287167, rgd1564463, a Kp3, ALAS2, gsg1, down-regulation of 10 genes was acot1, loc690226, hmgcs2, gbx1, retsat, klrb1b, ppp1r1c, slc27a2, loc683474, masp2. genes were screened the possible signal pathway related 25, as (1) biosynthesisofunsaturatedfattyacids (2) steroidbiosynthesis (3) synthesisanddegradationofketonebodies (4) glycosphingolipidbiosynthesis-globo (Series 5) PPAR signaling pathway (6) Fatty acid metabolism (7) Butanoate metabolism (8) Mineral absorption (9) Valine, leucine and isoleucine degradation (10) Steroid hormone biosynthesis (11) Ascorbate and aldarate metabolism (12) Porphyrin and chlorophyll metabolism (13) Glycosphingolipid biosynthesis-lacto and neolacto series (Retinol metabolism (14) 15) ABC transporters (16) Pentose and glucuronate interconversions (17) Drug metabolismcytochrome P450 -Rattus norvegicus (rat) (18) Drug metabolism-other enzymes-Rattus norvegicus (rat) (19) Metabolism of xenobiotics by cytochrome P450-Rattus norvegicus (rat) (20) Carbohydrate digestion and absorption (21) Peroxisome-Rattus norvegicus (rat) (22) Fat digestion and absorption (23) Starch and sucrose metabolism acid metabolism (Arachidonic (24) 25) Glycerophospholipid metabolism. 8 signaling pathways related to lipid metabolism; a total of 7 signal pathways and carbohydrate metabolism, polysaccharide; a total of 3 signal pathways and hormones, enzymes related to metabolism; a total of 2 signal pathways and intracellular transport, metabolism, cell membrane and other related activities; 1 signal pathways and cell proliferation, differentiation and other related activities. Conclusion: acupuncture can improve FD rats food intake and body weight, improve FD rat intestinal Gao Min disease, is the ideal treatment of FD Treatment; in FD rats, interstitial cells of Cajal and the number of abnormal morphology significantly decreased, which may be the cause of gastrointestinal motor dysfunction in FD rats. In FD rats, there are low immune function and immune dysfunction, which may be the cause of FD visceral hypersensitivity disease; acupuncture foot three points on show the treatment effect of FD better, suggesting that the treatment mechanism of acupuncture Zusanli can improve FD rat gastrointestinal tract injury and improve the quality of Cajal rat FD cells on immune function; molecular mechanism of FD disease, the screening of signaling pathway and the immune response, mast cell response, cell proliferation and differentiation, can be combined with differential gene and specific signaling pathways, to further deepen the research. Molecular mechanisms of acupuncture in treating FD disease at Zusanli, selected pathway and nutrition metabolism, absorption, secretion of the hormone regulation, can To further deepen the study in combination with related differential genes and specific signaling pathways.

【學(xué)位授予單位】：大連醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R245

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