慢性便秘患者肠道菌群对小鼠肠道5-羟色胺转运体表达及排便功能的影响
发布时间：2018-08-03 10:09
【摘要】：目的慢性便秘(chronic constipation)是一种常见下消化道疾病,表现为排便次数减少、粪便干结及排便困难,病程至少6个月。慢性便秘是常见的功能性胃肠病,人群发病率较高,症状持续反复,严重影响患者的身心健康及生活质量。慢性便秘的发病机制尚不清楚,近年来,已有研究表明慢性便秘患者的肠道菌群数量和种类较正常健康人有所改变,并且应用抗生素和微生态制剂可以缓解便秘症状,但其机制尚未明确。5-羟色胺(5-hydroxy tryptamine,5-HT)是脑一肠轴的关键神经递质,人体90%以上的5-HT由肠道合成。肠道合成的5-HT调节多种功能,包括肠道分泌、肠道运动和胃肠感觉功能。5-羟色胺转运体(serotonin transporte,SERT)是一种跨膜转运蛋白,广泛分布于肠道嗜铬细胞(enterochromaffin cells,EC cells)及神经突触前膜上,可将效应部位过多聚集的5-羟色胺再摄取,从而终止5-HT的作用。5-羟色胺和5-羟色胺转运体在慢性便秘的发病中有着重要的作用。研究表明,肠道微生态环境的改变可影响SERT的表达。本研究采用慢性便秘患者肠道菌群处理小鼠,评价对小鼠排便功能及肠道SERT表达的影响,旨在探讨肠道菌群失调在慢性便秘发生过程中的作用及可能的机制。方法本研究首先收集符合罗马Ⅲ的诊断标准的慢性便秘病人和健康正常人的粪便,运用焦磷酸测序法分析肠道菌群。将20只6周龄SPF级雌性C57BL/6小鼠分为2组,每组10只,一组为对照组,给予混合5个健康正常人的粪菌液灌胃,另一组为实验组,给予混合5个慢性便秘患者的粪菌液灌胃。灌胃前连续给予3天链霉素25mg预处理构建伪无菌鼠模型,继而给予粪菌液灌胃,4-6日每日一次,之后隔日一次,共7次,每只鼠每次250ul(浓度为1:10 g/ml),总共饲养的持续时间为15天。分别于第7天和第15天检测首粒排黑便时间、2h排便粒数、2h粪便重量、2h粪便干重及粪便含水率;第15天每组5只小鼠行小肠推进实验[实验前一天实验小鼠禁食不禁水16h,每只小鼠依照重量给予印度墨汁(0.2 m L/10 g)灌胃,25min后处死小鼠,取小肠置于托盘上,测量小肠总长度及墨汁推进长度。小肠推进率=墨汁推进长度/小肠总长度×100%]。最后处死小鼠,取其结肠组织。分别应用Realtime-PCR和Western blot检测小鼠结肠组织SERT m RNA和SERT蛋白表达,应用ELISA及免疫荧光方法检测小鼠结肠组织5-HT的水平,观察慢性便秘患者肠道菌群对小鼠肠道组织SERT及5-HT水平的影响。结果1.慢性便秘患者肠道菌群的多样性比对照组明显降低,F/B值(Firmicutes/Bacteroidetes)比对照组降低。比较某些重要的菌属发现慢性便秘患者组肠道菌属中双歧杆菌属(Bifidobacterium)、肠球菌(Enterococcus)、梭形杆菌属(Fusobacterium)的比例升高,梭菌属(Clostridium)、乳酸杆菌属(Lactobacillus)、链球菌属(Streptococcus)、埃希-志贺菌属(Escherichia-Shigella)及脱硫弧菌属(Desulfovibrio)的比例降低。2.慢性便秘患者肠道菌群对小鼠排便功能的影响:第7天的排便功能评价显示,慢性便秘患者肠道菌群可使小鼠2h粪便重量显著减少(271.86±32.72 vs223.24±47.50,P0.05),而首粒黑便排便时间(79.19±12.66 vs 89.08±16.11,P㧐0.05)、2 h排便粒数(15.42±2.63 vs 13.55±3.12,P㧐0.05)、2h粪便干重(134.91±25.18 vs 114.82±29.96,P㧐0.05)及粪便含水率(50.62%±5.42%vs48.88%±6.08%,P㧐0.05)无显著变化。第15天的排便功能评价显示,慢性便秘患者肠道菌群可使小鼠首粒黑便排便时间显著延长(69.06±2.72 vs83.24±11.31,P0.05),2 h排便粒数显著减少(12.14±2.90 vs 8.55±1.83,P0.05),2h粪便重量显著减少(246.72±64.01 vs 151.90±32.42,P0.01),2h粪便干重显著减少(92.93±23.07 vs 65.52±11.76,P0.05),粪便含水率显著减少(61.95%±3.70%vs 56.63%±3.01%,P0.05)。3.按照小肠墨汁推进率是否为100%(墨汁是否到达回盲部)进行比较,结果显示实验组墨汁推进率为100%的例数显著低于对照组(对照组5只小鼠小肠墨汁全部到达回盲部,实验组5只小鼠中仅2只小鼠小肠墨汁到达回盲部,两组差异有统计学意义(5/5 vs 2/5,P0.05)。4.慢性便秘患者肠道菌群干预的实验组小鼠肠道组织SERT m RNA表达水平与健康正常人肠道菌群肠道菌群干预的对照组相比显著增加(P0.05);实验组SERT蛋白的表达水平比亦明显高于对照组(P0.05)。5.慢性便秘患者肠道菌群干预的实验组小鼠肠道组织5-HT表达水平比健康正常人肠道菌群肠道菌群干预对照组显著降低(198.77±25.99 vs 151.69±10.18,P0.05);免疫荧光双染结果显示:与对照组相比,实验组小鼠肠道组织中5-HT的阳性表达明显减少。结论慢性便秘患者的肠道菌群能够增加小鼠肠道组织5-羟色胺转运体表达从而影响小鼠排便功能。肠道菌群失调可能在慢性便秘的发生过程中起到重要作用。
[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.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R574.62
本文编号：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.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R574.62
【参考文献】
相关期刊论文 前2条
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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