针刺足三里穴治疗功能性消化不良大鼠实验研究

发布时间：2018-01-01 08:23

本文关键词：针刺足三里穴治疗功能性消化不良大鼠实验研究　出处：《大连医科大学》2016年博士论文　论文类型：学位论文

【摘要】：背景:功能性消化不良(functional dyspepsia,FD)是临床最常见的一种功能性胃肠病,是指具有上腹痛、上腹胀、早饱、嗳气、食欲不振、恶心、呕吐等不适症状,经检查排除引起上述症状的器质性疾病的一组临床综合征,与中医的脾气虚证类似。症状可持续或反复发作,严重影响患者的生活质量,降低工作效率,增加了医疗资源的消耗。研究发现,FD患者存在胃肠道的动力异常及内脏高敏症,但该病的确切发病机制目前仍不清楚。针刺作为中国传统医学的一部分,在我国应用已有千余年的历史。足三里穴定位为犊鼻下3寸,胫骨前嵴外1横指处。中西医结合临床实践中,发现针刺足阳明胃经在功能性胃肠疾病方面具有自己独特的优势,尤其是针刺足三里穴应用较为广泛。很多研究均显示,针刺足三里穴可以有效缓解功能性胃肠病,对其确切作用机制,目前亦不完全清楚。目的:通过水浸法建立FD大鼠模型,观察大鼠日均摄食量、体重、肠道敏感性、小肠Cajal间质细胞、血清免疫指标的变化,通过表达谱基因芯片检测差异基因、差异基因的分子功能、差异基因参与的信号通路,进一步研究FD的发病机制。对FD大鼠进行针刺足三里穴治疗,观察大鼠日均摄食量、体重、肠道敏感性、小肠Cajal间质细胞、血清免疫指标的变化,通过表达谱基因芯片检测差异基因、差异基因的分子功能、差异基因参与的信号通路,探讨针刺足三里穴治疗FD疾病的机制。方法:成年Wistar大鼠被分成对照(control)组、模型(model)组、针刺足三里穴(acupuncture,AP)组、非经非穴针刺(sham acupuncture,sham-AP)组。除了control组,将其它组的大鼠通过水浸法建立FD模型。建立FD模型后,监测control组与model组大鼠摄食量、体重,评估大鼠肠道敏感性。通过结直肠扩张(colorectal distention,CRD)来诱发急性内脏痛,用大鼠对急性内脏痛的腹部回撤反射(abdominal withdrawal reflex,AWR)来评价肠道敏感性。取大鼠小肠组织,进行免疫荧光染色,共聚焦显微镜检测深肌层cajal间质细胞形态及数量。检测血清igg、igm、il-2和il-6水平。然后组间结果进行比较。取大鼠小肠组织,进行基因表达谱芯片检测,筛选model组与control组的差异基因,查找其分子功能,筛选差异基因相关信号通路。建立fd模型后,ap组大鼠针刺足三里穴15天,每天一次,每次时间为20分钟。sham-ap组大鼠于非经非穴点针刺15天,对model组大鼠进行束缚但不予针刺。对control组大鼠自然饲养。监测大鼠摄食量、体重,评估大鼠肠道敏感性。取大鼠小肠组织,进行免疫荧光染色,共聚焦显微镜检测深肌层cajal间质细胞形态及数量。检测血清igg、igm、il-2和il-6水平。然后将control组、model组、ap组和sham-ap组间数据进行比较。取大鼠小肠组织,进行基因表达谱芯片检测,筛选ap组与model组的差异基因,查找其分子功能,筛选差异基因相关信号通路。结果:1.model组大鼠的日均摄食量明显低于control组(t=-5.360p0.01);体重明显低于control组(t=-3.144p0.05);肠道敏感性明显高于control组。在model组,当crd的压力为20、40、60或80mmhg时,awr评分均显著高于control组(t=5.00,p0.01;t=2.71,p0.05;t=2.71,p0.05;t=3.16,p0.05)。再经过针刺足三里穴15天后,ap组(针刺组)大鼠的日均摄食量明显高于model组(非针刺组)(p0.05);体重明显高于model组(p0.05);当crd的压力为20mmhg时,ap组大鼠的肠道高敏症较model组无明显差异(p0.05)。当crd的压力为40、60或80mmhg时,ap组大鼠的肠道高敏症较model组减轻(p0.05,p0.05,p0.05)。2.model组大鼠的小肠cajal间质细胞形态明显不正常且数量低于control组。cajal间质细胞的免疫荧光值显著低于control组(t=-21.45,p0.01)。再经过针刺足三里穴15天后,ap组大鼠的小肠cajal间质细胞形态恢复且数量明显高于model组。cajal间质细胞的免疫荧光值显著高于model组(p0.05)。3.model组大鼠的血清igg、igm和il-2水平明显低于control组(t=-11.67,p0.01;t=-11.26,p0.01;t=-4.44,p0.01),血清il-6水平明显高于control组(t=5.23,p0.01)。再经过针刺足三里穴15天后,ap组大鼠的血清igg、igm和il-2水平明显高于model组(p0.05;p0.05;p0.05),血清il-6水平明显低于model组(p0.05)。4.小肠组织基因表达谱芯片检测:排除无geneid的基因,以差异倍数(foldchange,fc)2或0.5,p0.01为标准,model组大鼠和control组相比,筛选差异基因,有255个差异基因,其中上调基因11个,下调基因244个。上调最显著的10个基因是rgd1559622、tsks、gramd2、colq、kcnq4、mecom、tulp1、thrb、acot1、lss,下调最显著的10基因是loc100362150、mcpt2、igh-6、mcpt1l4、cpa3、mcpt8l2、fcer1a、loc363827、gpr174、loc100363695。筛选出差异基因可能相关的信号通路19条,为(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条信号通路明显与免疫反应相关,其中3条信号通路与肥大细胞反应相关;共5条信号通路与细胞增殖、分化、凋亡相关;共2条信号通路与细胞内活动、细胞内代谢、细胞运动有关。ap组大鼠和model组相比,筛选差异基因,有169个差异基因,其中上调基因96个,下调基因73个。上调最显著的10个基因是slc34a2、trpv6、gucy2g、hbb、hbb-b1、loc287167、rgd1564463、akp3、alas2、gsg1,下调最显著的10个基因是acot1、loc690226、hmgcs2、gbx1、retsat、klrb1b、ppp1r1c、slc27a2、loc683474、masp2。筛选出差异基因可能相关的信号通路25条,为(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条信号通路与脂类代谢有关;共7条信号通路与碳水化合物、多糖代谢有关;共3条信号通路与激素、酶等代谢有关;共2条信号通路与细胞内转运、分解代谢,细胞膜活动等相关;1条信号通路与细胞增殖、分化等活动相关。结论:针刺足三里穴能够提高FD大鼠摄食量和体重,改善FD大鼠肠道高敏症,是治疗FD的理想治疗方式;在FD大鼠,小肠Cajal间质细胞形态异常且数量明显降低,这可能是FD大鼠胃肠运动功能障碍的原因。在FD大鼠,存在免疫功能低下及免疫功能失调,这可能是FD内脏高敏症的原因;针刺足三里穴显示出对FD较好的治疗效果,提示治疗的机制可能与针刺足三里穴能够改善FD大鼠胃肠道Cajal间质细胞损伤和改善FD大鼠免疫功能有关;FD疾病的分子机制方面,所筛选信号通路与免疫反应,肥大细胞反应,细胞增殖分化等方面相关,可结合相关差异基因及具体信号通路,进一步深化研究。针刺足三里穴治疗FD疾病的分子机制方面,所筛选信号通路与营养代谢调节、吸收,激素分泌调节等相关,可结合相关的差异基因及具体信号通路,进一步深化研究。
[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.

【学位授予单位】：大连医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R245

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