慢性便秘患者腸道菌群對(duì)小鼠腸道5-羥色胺轉(zhuǎn)運(yùn)體表達(dá)及排便功能的影響
發(fā)布時(shí)間：2018-08-03 10:09
【摘要】：目的慢性便秘(chronic constipation)是一種常見下消化道疾病,表現(xiàn)為排便次數(shù)減少、糞便干結(jié)及排便困難,病程至少6個(gè)月。慢性便秘是常見的功能性胃腸病,人群發(fā)病率較高,癥狀持續(xù)反復(fù),嚴(yán)重影響患者的身心健康及生活質(zhì)量。慢性便秘的發(fā)病機(jī)制尚不清楚,近年來,已有研究表明慢性便秘患者的腸道菌群數(shù)量和種類較正常健康人有所改變,并且應(yīng)用抗生素和微生態(tài)制劑可以緩解便秘癥狀,但其機(jī)制尚未明確。5-羥色胺(5-hydroxy tryptamine,5-HT)是腦一腸軸的關(guān)鍵神經(jīng)遞質(zhì),人體90%以上的5-HT由腸道合成。腸道合成的5-HT調(diào)節(jié)多種功能,包括腸道分泌、腸道運(yùn)動(dòng)和胃腸感覺功能。5-羥色胺轉(zhuǎn)運(yùn)體(serotonin transporte,SERT)是一種跨膜轉(zhuǎn)運(yùn)蛋白,廣泛分布于腸道嗜鉻細(xì)胞(enterochromaffin cells,EC cells)及神經(jīng)突觸前膜上,可將效應(yīng)部位過多聚集的5-羥色胺再攝取,從而終止5-HT的作用。5-羥色胺和5-羥色胺轉(zhuǎn)運(yùn)體在慢性便秘的發(fā)病中有著重要的作用。研究表明,腸道微生態(tài)環(huán)境的改變可影響SERT的表達(dá)。本研究采用慢性便秘患者腸道菌群處理小鼠,評(píng)價(jià)對(duì)小鼠排便功能及腸道SERT表達(dá)的影響,旨在探討腸道菌群失調(diào)在慢性便秘發(fā)生過程中的作用及可能的機(jī)制。方法本研究首先收集符合羅馬Ⅲ的診斷標(biāo)準(zhǔn)的慢性便秘病人和健康正常人的糞便,運(yùn)用焦磷酸測(cè)序法分析腸道菌群。將20只6周齡SPF級(jí)雌性C57BL/6小鼠分為2組,每組10只,一組為對(duì)照組,給予混合5個(gè)健康正常人的糞菌液灌胃,另一組為實(shí)驗(yàn)組,給予混合5個(gè)慢性便秘患者的糞菌液灌胃。灌胃前連續(xù)給予3天鏈霉素25mg預(yù)處理構(gòu)建偽無菌鼠模型,繼而給予糞菌液灌胃,4-6日每日一次,之后隔日一次,共7次,每只鼠每次250ul(濃度為1:10 g/ml),總共飼養(yǎng)的持續(xù)時(shí)間為15天。分別于第7天和第15天檢測(cè)首粒排黑便時(shí)間、2h排便粒數(shù)、2h糞便重量、2h糞便干重及糞便含水率;第15天每組5只小鼠行小腸推進(jìn)實(shí)驗(yàn)[實(shí)驗(yàn)前一天實(shí)驗(yàn)小鼠禁食不禁水16h,每只小鼠依照重量給予印度墨汁(0.2 m L/10 g)灌胃,25min后處死小鼠,取小腸置于托盤上,測(cè)量小腸總長度及墨汁推進(jìn)長度。小腸推進(jìn)率=墨汁推進(jìn)長度/小腸總長度×100%]。最后處死小鼠,取其結(jié)腸組織。分別應(yīng)用Realtime-PCR和Western blot檢測(cè)小鼠結(jié)腸組織SERT m RNA和SERT蛋白表達(dá),應(yīng)用ELISA及免疫熒光方法檢測(cè)小鼠結(jié)腸組織5-HT的水平,觀察慢性便秘患者腸道菌群對(duì)小鼠腸道組織SERT及5-HT水平的影響。結(jié)果1.慢性便秘患者腸道菌群的多樣性比對(duì)照組明顯降低,F/B值(Firmicutes/Bacteroidetes)比對(duì)照組降低。比較某些重要的菌屬發(fā)現(xiàn)慢性便秘患者組腸道菌屬中雙歧桿菌屬(Bifidobacterium)、腸球菌(Enterococcus)、梭形桿菌屬(Fusobacterium)的比例升高,梭菌屬(Clostridium)、乳酸桿菌屬(Lactobacillus)、鏈球菌屬(Streptococcus)、埃希-志賀菌屬(Escherichia-Shigella)及脫硫弧菌屬(Desulfovibrio)的比例降低。2.慢性便秘患者腸道菌群對(duì)小鼠排便功能的影響:第7天的排便功能評(píng)價(jià)顯示,慢性便秘患者腸道菌群可使小鼠2h糞便重量顯著減少(271.86±32.72 vs223.24±47.50,P0.05),而首粒黑便排便時(shí)間(79.19±12.66 vs 89.08±16.11,P㧐0.05)、2 h排便粒數(shù)(15.42±2.63 vs 13.55±3.12,P㧐0.05)、2h糞便干重(134.91±25.18 vs 114.82±29.96,P㧐0.05)及糞便含水率(50.62%±5.42%vs48.88%±6.08%,P㧐0.05)無顯著變化。第15天的排便功能評(píng)價(jià)顯示,慢性便秘患者腸道菌群可使小鼠首粒黑便排便時(shí)間顯著延長(69.06±2.72 vs83.24±11.31,P0.05),2 h排便粒數(shù)顯著減少(12.14±2.90 vs 8.55±1.83,P0.05),2h糞便重量顯著減少(246.72±64.01 vs 151.90±32.42,P0.01),2h糞便干重顯著減少(92.93±23.07 vs 65.52±11.76,P0.05),糞便含水率顯著減少(61.95%±3.70%vs 56.63%±3.01%,P0.05)。3.按照小腸墨汁推進(jìn)率是否為100%(墨汁是否到達(dá)回盲部)進(jìn)行比較,結(jié)果顯示實(shí)驗(yàn)組墨汁推進(jìn)率為100%的例數(shù)顯著低于對(duì)照組(對(duì)照組5只小鼠小腸墨汁全部到達(dá)回盲部,實(shí)驗(yàn)組5只小鼠中僅2只小鼠小腸墨汁到達(dá)回盲部,兩組差異有統(tǒng)計(jì)學(xué)意義(5/5 vs 2/5,P0.05)。4.慢性便秘患者腸道菌群干預(yù)的實(shí)驗(yàn)組小鼠腸道組織SERT m RNA表達(dá)水平與健康正常人腸道菌群腸道菌群干預(yù)的對(duì)照組相比顯著增加(P0.05);實(shí)驗(yàn)組SERT蛋白的表達(dá)水平比亦明顯高于對(duì)照組(P0.05)。5.慢性便秘患者腸道菌群干預(yù)的實(shí)驗(yàn)組小鼠腸道組織5-HT表達(dá)水平比健康正常人腸道菌群腸道菌群干預(yù)對(duì)照組顯著降低(198.77±25.99 vs 151.69±10.18,P0.05);免疫熒光雙染結(jié)果顯示:與對(duì)照組相比,實(shí)驗(yàn)組小鼠腸道組織中5-HT的陽性表達(dá)明顯減少。結(jié)論慢性便秘患者的腸道菌群能夠增加小鼠腸道組織5-羥色胺轉(zhuǎn)運(yùn)體表達(dá)從而影響小鼠排便功能。腸道菌群失調(diào)可能在慢性便秘的發(fā)生過程中起到重要作用。
[Abstract]:Objective chronic constipation (chronic constipation) is a common lower digestive tract disease, which shows a decrease in the number of defecation, difficult stool knot and defecation, and at least 6 months. Chronic constipation is a common functional gastrointestinal disease. The incidence of chronic constipation is high, the symptoms are repeated, severe weight affects the physical and mental health and quality of life of the patients. Chronic constipation The pathogenesis is not clear. In recent years, studies have shown that the number and type of intestinal flora in patients with chronic constipation have been changed more than normal healthy people, and the application of antibiotics and microecological agents can relieve constipation symptoms, but the mechanism has not yet been identified as the key neurotransmitter of.5- serotonin (5-hydroxy tryptamine, 5-HT). More than 90% of the body of 5-HT is synthesized by the intestinal tract. The 5-HT regulates a variety of functions, including intestinal secretion, intestinal motility and gastrointestinal sensory function,.5- serotonin transporter (serotonin transporte, SERT), a transmembrane transporter, widely distributed in the intestinal chromaffin cells (enterochromaffin cells, EC cells) and the nerve presynaptic membrane. The effect of 5- HT reuptake, which terminates 5-HT, terminates the role of.5- serotonin and 5- serotonin transporter in the pathogenesis of chronic constipation. The study shows that the changes in the intestinal microecological environment can affect the expression of SERT. This study used the intestinal flora of patients with chronic constipation to treat mice and evaluate the mouse platoon. The effect of stool function and the expression of SERT in the intestinal tract was designed to explore the role and possible mechanism of intestinal flora imbalance in the process of chronic constipation. Methods firstly, the feces of chronic constipation patients and healthy people who were in accordance with the diagnostic criteria of Rome III were collected, and the intestinal flora was analyzed by pyrophosphoric acid sequencing method. 20 6 weeks of age of SPF grade female were analyzed. The sex C57BL/6 mice were divided into 2 groups, each group was 10. One group was the control group, and 5 healthy normal people were given the fecal bacteria. The other group was treated with fecal bacteria in 5 chronic constipation patients. Before gavage, the pseudosterilized rat model of streptomycin 25mg preconditioning was continuously given for 3 days, then the fecal bacteria were given to the stomach. The 4-6 day was a daily one. A total of 7 times, 7 times every other day, each rat was 250ul at a concentration of 1:10 g/ml, and the duration of the total feeding was 15 days. The time of black stool, the number of 2H defecation, the weight of 2H feces, the dry weight of 2H stool and the water content of feces were measured on the seventh and fifteenth days, respectively, and 5 mice in each group were subjected to small intestinal propulsion experiment on the fifteenth day of the experiment [experimental mice a day before the experiment. The fasting could not help water 16h, each mouse was given the India ink (0.2 m L/10 g) according to the weight of the stomach. After 25min, the mice were killed and the small intestine was placed on the tray to measure the length of the small intestine and the length of the ink. The small intestine propulsive rate = the length of the ink / the total length of the small intestine and the length of the small intestine were killed in the mice, and the colon tissue was taken. Realtime-PCR and Weste were applied respectively. The expression of SERT m RNA and SERT protein in the colon tissue of mice was detected by RN blot. The level of 5-HT in the colon tissue of mice was detected by ELISA and immunofluorescence. The effect of intestinal microflora on the SERT and 5-HT levels of intestinal tissue in mice with chronic constipation was observed. Results 1. the diversity of intestinal flora in the patients with chronic constipation was significantly lower than that in the control group, and the F/B (Fi) value (Fi) was significantly lower than that of the control group. Rmicutes/Bacteroidetes) lower than the control group. Some important bacteria were found in the group of chronic constipation in the group of intestinal bacteria in the genus Bifidobacterium (Bifidobacterium), Enterococcus (Enterococcus), Clostridium (Fusobacterium), Clostridium (Clostridium), Lactobacillus (Lactobacillus), Streptococcus (Streptococcus), and the genus Streptococcus (Streptococcus). The proportion of Escherichia-Shigella and Desulfovibrio decreased the effect of intestinal microflora in.2. patients with chronic constipation. The seventh day defecation evaluation showed that the intestinal flora of patients with chronic constipation could significantly reduce the weight of 2H feces in mice (271.86 + 32.72 vs223.24 47.50, P0.05). The time of fecal defecation (79.19 + 12.66 vs 89.08 + 16.11, P? 0.05), 2 h defecation particles (15.42 + 2.63 vs 13.55 + 3.12, P? 0.05), 2h fecal dry weight (134.91 + 25.18 vs 114.82 + 2, P? P?) and fecal moisture content (P? P?) showed that the bowel bacteria in chronic constipation patients showed intestinal bacteria in chronic constipation The time of fecal defecation was significantly prolonged (69.06 + 2.72 vs83.24 + 11.31, P0.05), and the number of fecal particles in 2 h decreased significantly (12.14 + 2.90 vs 8.55 + 1.83, P0.05), and the weight of 2H stool decreased significantly (246.72 + 64.01 vs 151.90 + 32.42, P0.01), and the dry weight of 2H excrement decreased significantly (92.93 + vs, vs, P0.05), and the water content of feces decreased significantly Less (61.95% + 3.70%vs 56.63% + 3.01%, P0.05).3. was compared with whether the propelling rate of small intestine ink was 100% (whether ink reached the ileocecum). The results showed that the number of ink propellant rate of 100% in the experimental group was significantly lower than that of the control group (5 mice in the control group were all to the blind part of the small intestine, and only 2 mice in the experimental group had only 2 mice in the small intestine ink. " To the ileocecum, the two groups were statistically significant (5/5 vs 2/5, P0.05).4. chronic constipation patients with intestinal microflora intervention in the experimental group, the expression level of SERT m RNA in the intestinal tissue of the mice was significantly higher than that in the control group (P0.05), and the expression level of SERT protein in the experimental group was significantly higher than that of the control group (P0.05). Group (P0.05).5. chronic constipation patients with intestinal microflora intervention in the experimental group of mouse intestinal tissue 5-HT expression level was significantly lower than the healthy normal intestinal microflora intervention control group (198.77 + 25.99 vs 151.69 + 10.18, P0.05); immunofluorescence double staining results showed that compared with the experimental group, the positive table of 5-HT in the intestinal tissue of the experimental group mice Conclusion the intestinal microflora in patients with chronic constipation can increase the expression of 5- serotonin transporter in the intestinal tissue of mice and affect the function of mouse defecation. Intestinal flora imbalance may play an important role in the process of chronic constipation.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R574.62
本文編號(hào)：2161412
[Abstract]:Objective chronic constipation (chronic constipation) is a common lower digestive tract disease, which shows a decrease in the number of defecation, difficult stool knot and defecation, and at least 6 months. Chronic constipation is a common functional gastrointestinal disease. The incidence of chronic constipation is high, the symptoms are repeated, severe weight affects the physical and mental health and quality of life of the patients. Chronic constipation The pathogenesis is not clear. In recent years, studies have shown that the number and type of intestinal flora in patients with chronic constipation have been changed more than normal healthy people, and the application of antibiotics and microecological agents can relieve constipation symptoms, but the mechanism has not yet been identified as the key neurotransmitter of.5- serotonin (5-hydroxy tryptamine, 5-HT). More than 90% of the body of 5-HT is synthesized by the intestinal tract. The 5-HT regulates a variety of functions, including intestinal secretion, intestinal motility and gastrointestinal sensory function,.5- serotonin transporter (serotonin transporte, SERT), a transmembrane transporter, widely distributed in the intestinal chromaffin cells (enterochromaffin cells, EC cells) and the nerve presynaptic membrane. The effect of 5- HT reuptake, which terminates 5-HT, terminates the role of.5- serotonin and 5- serotonin transporter in the pathogenesis of chronic constipation. The study shows that the changes in the intestinal microecological environment can affect the expression of SERT. This study used the intestinal flora of patients with chronic constipation to treat mice and evaluate the mouse platoon. The effect of stool function and the expression of SERT in the intestinal tract was designed to explore the role and possible mechanism of intestinal flora imbalance in the process of chronic constipation. Methods firstly, the feces of chronic constipation patients and healthy people who were in accordance with the diagnostic criteria of Rome III were collected, and the intestinal flora was analyzed by pyrophosphoric acid sequencing method. 20 6 weeks of age of SPF grade female were analyzed. The sex C57BL/6 mice were divided into 2 groups, each group was 10. One group was the control group, and 5 healthy normal people were given the fecal bacteria. The other group was treated with fecal bacteria in 5 chronic constipation patients. Before gavage, the pseudosterilized rat model of streptomycin 25mg preconditioning was continuously given for 3 days, then the fecal bacteria were given to the stomach. The 4-6 day was a daily one. A total of 7 times, 7 times every other day, each rat was 250ul at a concentration of 1:10 g/ml, and the duration of the total feeding was 15 days. The time of black stool, the number of 2H defecation, the weight of 2H feces, the dry weight of 2H stool and the water content of feces were measured on the seventh and fifteenth days, respectively, and 5 mice in each group were subjected to small intestinal propulsion experiment on the fifteenth day of the experiment [experimental mice a day before the experiment. The fasting could not help water 16h, each mouse was given the India ink (0.2 m L/10 g) according to the weight of the stomach. After 25min, the mice were killed and the small intestine was placed on the tray to measure the length of the small intestine and the length of the ink. The small intestine propulsive rate = the length of the ink / the total length of the small intestine and the length of the small intestine were killed in the mice, and the colon tissue was taken. Realtime-PCR and Weste were applied respectively. The expression of SERT m RNA and SERT protein in the colon tissue of mice was detected by RN blot. The level of 5-HT in the colon tissue of mice was detected by ELISA and immunofluorescence. The effect of intestinal microflora on the SERT and 5-HT levels of intestinal tissue in mice with chronic constipation was observed. Results 1. the diversity of intestinal flora in the patients with chronic constipation was significantly lower than that in the control group, and the F/B (Fi) value (Fi) was significantly lower than that of the control group. Rmicutes/Bacteroidetes) lower than the control group. Some important bacteria were found in the group of chronic constipation in the group of intestinal bacteria in the genus Bifidobacterium (Bifidobacterium), Enterococcus (Enterococcus), Clostridium (Fusobacterium), Clostridium (Clostridium), Lactobacillus (Lactobacillus), Streptococcus (Streptococcus), and the genus Streptococcus (Streptococcus). The proportion of Escherichia-Shigella and Desulfovibrio decreased the effect of intestinal microflora in.2. patients with chronic constipation. The seventh day defecation evaluation showed that the intestinal flora of patients with chronic constipation could significantly reduce the weight of 2H feces in mice (271.86 + 32.72 vs223.24 47.50, P0.05). The time of fecal defecation (79.19 + 12.66 vs 89.08 + 16.11, P? 0.05), 2 h defecation particles (15.42 + 2.63 vs 13.55 + 3.12, P? 0.05), 2h fecal dry weight (134.91 + 25.18 vs 114.82 + 2, P? P?) and fecal moisture content (P? P?) showed that the bowel bacteria in chronic constipation patients showed intestinal bacteria in chronic constipation The time of fecal defecation was significantly prolonged (69.06 + 2.72 vs83.24 + 11.31, P0.05), and the number of fecal particles in 2 h decreased significantly (12.14 + 2.90 vs 8.55 + 1.83, P0.05), and the weight of 2H stool decreased significantly (246.72 + 64.01 vs 151.90 + 32.42, P0.01), and the dry weight of 2H excrement decreased significantly (92.93 + vs, vs, P0.05), and the water content of feces decreased significantly Less (61.95% + 3.70%vs 56.63% + 3.01%, P0.05).3. was compared with whether the propelling rate of small intestine ink was 100% (whether ink reached the ileocecum). The results showed that the number of ink propellant rate of 100% in the experimental group was significantly lower than that of the control group (5 mice in the control group were all to the blind part of the small intestine, and only 2 mice in the experimental group had only 2 mice in the small intestine ink. " To the ileocecum, the two groups were statistically significant (5/5 vs 2/5, P0.05).4. chronic constipation patients with intestinal microflora intervention in the experimental group, the expression level of SERT m RNA in the intestinal tissue of the mice was significantly higher than that in the control group (P0.05), and the expression level of SERT protein in the experimental group was significantly higher than that of the control group (P0.05). Group (P0.05).5. chronic constipation patients with intestinal microflora intervention in the experimental group of mouse intestinal tissue 5-HT expression level was significantly lower than the healthy normal intestinal microflora intervention control group (198.77 + 25.99 vs 151.69 + 10.18, P0.05); immunofluorescence double staining results showed that compared with the experimental group, the positive table of 5-HT in the intestinal tissue of the experimental group mice Conclusion the intestinal microflora in patients with chronic constipation can increase the expression of 5- serotonin transporter in the intestinal tissue of mice and affect the function of mouse defecation. Intestinal flora imbalance may play an important role in the process of chronic constipation.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R574.62
【參考文獻(xiàn)】
相關(guān)期刊論文 前2條
1 Xiu-Fang Cui;Wei-Mei Zhou;Yan Yang;Jun Zhou;Xue-Liang Li;Lin Lin;Hong-Jie Zhang;;Epidermal growth factor upregulates serotonin transporter and its association with visceral hypersensitivity in irritable bowel syndrome[J];World Journal of Gastroenterology;2014年37期
2 Anna Chmielewska;Hania Szajewska;;Systematic review of randomised controlled trials:Probiotics for functional constipation[J];World Journal of Gastroenterology;2010年01期
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