人体不同节段小肠腔菌群与膜菌群的研究
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摘要
背景与研究目的
近年来研究发现，小肠疾病并非少见，尤其是糜烂性和溃疡性疾病更为常见，内镜下取材活检多为慢性炎症，不能得到明确的诊断，小肠的这类疾病是否属感染性或非感染性?小肠的不同节段微生态状况如何?在小肠疾病的发生中发挥什么样的作用?这些都是不明确的问题。目前国内外对于肠道微生态学的研究主要集中在：肠道微生态与肠黏膜屏障功能衰竭的关系、肠道微生态与炎症性肠病的关系、肠道菌群的分子生物学分析等。随着分子生物学等多种新技术的进步与普及，促进了微生态学研究的深入，其涉及的领域包括肠道菌及其产物在粘膜免疫发育与效用中的作用、应用肠固有菌群作为疫苗或基因治疗的载体菌等。由于内镜取材的限止目前研究主要是空肠始段和回肠末段的微生态学，关于全小肠不同节段的肠道腔菌群与膜菌群的研究未见报道。本研究通过对全小肠不同节段的肠道腔菌群与膜菌群的研究，揭示生理情况下小肠菌群的组成和分布特点，为进一步研究小肠微生态在小肠疾病中的作用提供参考。
对象和方法
研究对象：13例非腹部外伤的意外伤亡者。
目的菌：对10种肠道腔菌群与膜菌群的微生态情况进行分析，目的菌为4种需氧菌(肠杆菌、肠球菌、葡萄球菌及酵母菌)和6种厌氧菌(拟杆菌、乳杆菌、梭菌、双歧杆菌、真杆菌和消化链球菌)。
标本的选取：以受检者小肠的空肠上段、空肠中段、空回交界处、回肠中段、回肠末段为取材点。
方法：腔菌群的滴种：在相对低温(避免细菌增殖而影响数据)的环境下按倍比稀释的方法取0.1ml含腔内容物+0.9ml稀释液配成第1EP管，按10~(-1)10~(-2)10~(-3)10~(-4)10~(-5)10~(-6)顺序依次稀释，滴种于预先制备好的10种选择性培养基上，每个平板接种6个稀释度，一个稀释度滴3滴，分送需氧及厌氧培养，然后计菌
Background & Objectives: Diseases of small intestine have been frequently found over the past few years, especially the anabrotic and ulcerative diseases.These diseases cannot be diagnosed with certainty by endoscopy and biopsy ,which only hint chronic inflammation. Whether these diseases of small intestine are infective,what the condition is and to what contribution of microbial population in different small intestinal lumen are still not clear.The recent studies mainly focus on the relationship of microbial population and dysfunction of enteric membrane , the relationship of microbial population and inflammatory bowel disease(IBD),the analysis of microbial population by molecular biology technology.However, the extent of these studies are only limited to the distal ileum ,so it is necessary to study the microbial population in different small intestinal lumen and in different enteric membrane to reveal the composition and distribution of intestinal microbial population under physiological functions.It will facilitate the research of effect of microbial population on intestinal diseases.
Materials and Methods
Materials: superior segment of jejunum,middle segment of jejunum junction domain of jejunum and ileum,middle segment of ileum,distal ileum(From 13 people dying of accident without abdominal injury)
Objective bacteriums: Oxybiontic
bacteria:Entericbacilli(EB),Enterococcus(EC),Staphylcocci(SP), Yeast fungus(YF);Anaerobicbacterium:Bacteroid(Bd),Bacillusbifidus(BL),Peptostreptococc us(PS),Lactobacilli(LC),Eubacterium(ES),Clostridium(CD)
Methods:
近年来研究发现，小肠疾病并非少见，尤其是糜烂性和溃疡性疾病更为常见，内镜下取材活检多为慢性炎症，不能得到明确的诊断，小肠的这类疾病是否属感染性或非感染性?小肠的不同节段微生态状况如何?在小肠疾病的发生中发挥什么样的作用?这些都是不明确的问题。目前国内外对于肠道微生态学的研究主要集中在：肠道微生态与肠黏膜屏障功能衰竭的关系、肠道微生态与炎症性肠病的关系、肠道菌群的分子生物学分析等。随着分子生物学等多种新技术的进步与普及，促进了微生态学研究的深入，其涉及的领域包括肠道菌及其产物在粘膜免疫发育与效用中的作用、应用肠固有菌群作为疫苗或基因治疗的载体菌等。由于内镜取材的限止目前研究主要是空肠始段和回肠末段的微生态学，关于全小肠不同节段的肠道腔菌群与膜菌群的研究未见报道。本研究通过对全小肠不同节段的肠道腔菌群与膜菌群的研究，揭示生理情况下小肠菌群的组成和分布特点，为进一步研究小肠微生态在小肠疾病中的作用提供参考。
对象和方法
研究对象：13例非腹部外伤的意外伤亡者。
目的菌：对10种肠道腔菌群与膜菌群的微生态情况进行分析，目的菌为4种需氧菌(肠杆菌、肠球菌、葡萄球菌及酵母菌)和6种厌氧菌(拟杆菌、乳杆菌、梭菌、双歧杆菌、真杆菌和消化链球菌)。
标本的选取：以受检者小肠的空肠上段、空肠中段、空回交界处、回肠中段、回肠末段为取材点。
方法：腔菌群的滴种：在相对低温(避免细菌增殖而影响数据)的环境下按倍比稀释的方法取0.1ml含腔内容物+0.9ml稀释液配成第1EP管，按10~(-1)10~(-2)10~(-3)10~(-4)10~(-5)10~(-6)顺序依次稀释，滴种于预先制备好的10种选择性培养基上，每个平板接种6个稀释度，一个稀释度滴3滴，分送需氧及厌氧培养，然后计菌
Background & Objectives: Diseases of small intestine have been frequently found over the past few years, especially the anabrotic and ulcerative diseases.These diseases cannot be diagnosed with certainty by endoscopy and biopsy ,which only hint chronic inflammation. Whether these diseases of small intestine are infective,what the condition is and to what contribution of microbial population in different small intestinal lumen are still not clear.The recent studies mainly focus on the relationship of microbial population and dysfunction of enteric membrane , the relationship of microbial population and inflammatory bowel disease(IBD),the analysis of microbial population by molecular biology technology.However, the extent of these studies are only limited to the distal ileum ,so it is necessary to study the microbial population in different small intestinal lumen and in different enteric membrane to reveal the composition and distribution of intestinal microbial population under physiological functions.It will facilitate the research of effect of microbial population on intestinal diseases.
Materials and Methods
Materials: superior segment of jejunum,middle segment of jejunum junction domain of jejunum and ileum,middle segment of ileum,distal ileum(From 13 people dying of accident without abdominal injury)
Objective bacteriums: Oxybiontic
bacteria:Entericbacilli(EB),Enterococcus(EC),Staphylcocci(SP), Yeast fungus(YF);Anaerobicbacterium:Bacteroid(Bd),Bacillusbifidus(BL),Peptostreptococc us(PS),Lactobacilli(LC),Eubacterium(ES),Clostridium(CD)
Methods:
引文
1、康白．人类胃肠道微生态学．见：康白．主编．微生态学．第1版．大连：大连出版社 1998：200-221．
2、张达容 微生态学在医学领域中的应用[J] 中国微生态学杂志 2002，2(14)3-4，
3、Steidler L, In situ delivery of cytokines by genetically engineered Lactococc us lactis [J] Antonie Van Leeu wenhoek, 2002,82(14):323-231
4. Gorbach SL, Plaut AG, Nahas L, et al, Studies of intestinal microflora. Gastroenterology, 1967, 53:856
5．潘令嘉等 正常国人小肠腔菌群及膜菌群的研究[J]中华内科杂志 1995，35(10)；686
6．潘令嘉等 小肠内容物腔菌群及膜菌群的研究[J]中国微生态学杂志1996，8(6)1-8，
7．熊得鑫．厌氧菌得分离和鉴定方法．南昌：江西科学技术出版社，1986．163-173
8．熊得鑫．使用悉生大鼠建立肠道，膜菌群的方法．中国微生态杂志，1993，5(2)：47
9 Molin G, Jeppsson B, Johansson ML,et al. Numerical taxonomyof different lactobacillus spp associated with healthy and diseasedmucosa of the human intestines. J Appl Bacteriol,1993,74 (3):314
10 Johansson ML, Molin G, Jeppsson B, et al. Administration of different lactobacillus strains in fermented oatmeal soup: in vivo colonization of human intestinal mucosa and effect on the indige2nous flora. Appl Environ Microbiol,1993,59 (1): 15
1 康白．人类胃肠道微生态学．见：康白．主编．微生态学．第1版．大连：大连出版社 1998：200-221．
2 康白．正常微生物群的生理代谢．见：康白．主编．微生态学．第1版．大连：大连出版社 1998：82-93．
3 康白．正常微生物群与其病变的相互关系．见：康白．主编．微生态学．第1版．大连：大连出版社 1998：64-80．
4 迟明，李颜．微生态制剂在儿童肠道疾病的应用进展．中国微生态学杂志2006；18：80．
5 Lee MC, Lin LH, Hung KL, et al. Oral bacterial therapy promotes recovery from acute diarrhea in children. Acta Paediatr Taiwan 2001;42:301-305.
6 黄柳革．金双歧、思密达联合治疗小儿急性腹泻病的疗效观察．中国微生态学杂志 2005；17：39-40．
7 董文岚．微生态制剂(金双歧)治疗婴幼儿腹泻60例临床观察．中国微生态学杂志 2005；17：40．
8 崔海宏，陈村龙，孙勇，等．溃疡性结肠炎患者肠黏膜菌群改变及抗体反应．中国微生态学杂志 2003；15：151-152．
9 Guslandi M, Mezzi G, Sorghi M, et al. Saccharomyces boulardii in maintenance treatment of Crohn's disease. Dig Dis Sci 2000;45:1462-1464.
10 Dunne C. Adaptation of bacteria to the intestinal niche: probiotics and gut disorder. Inflamm Bowel Dis 2001;7:136-145.
11 Bergogne-Berezin E. Treatment and prevention of antibiotic associ—ated diarrhea. Int J Antimicrob Agents 2000;16:521-526.
12 Olah A, Belagyi T, Issekutz A, et al. Randomized clinical trial of specific lactobacillus and fibre supplement to early enteral nutrition in patients with acute pancreatitis. Br J Surg 2002;89:1103-1107.
13 蒋虹，胡宏．双歧杆菌表面分子诱导大肠癌细胞分化的研究．中华微生物学和免疫学杂志 2001；21：186-190．
14 王立生，潘令嘉，陈村龙，等．双歧杆菌对裸鼠移植大肠癌增殖与凋亡的原位研究．世界华人消化杂志 2000；8：429-431．
15 Challa A, Rao DR, Chawan CB, et al. Bifidobacterium longum and lactulose suppress azoxymethane-indueed colonic aberrant crypt fo—ci in rats. Carcinogenesis 1997;18:517-521.
16 杨景云，杨继新．中医药微生态学的研究现状．中国微生态学杂志2003；15：1-3．
17 刘佳文，吕红艳．16SrRNA基因序列在分枝杆菌分类鉴定的研究进展．国外医学临床生物化学与检验学分册 2001；22：265-268．
18 Leblond-Bourget N, Philippe H, Mangin I, et al. 16S rRNA and 16S to 23S internal transcribed spacer sequence analyses reveal inter- and intraspecific Bifidobacterium phylogeny. Int J Syst Bacteriol 1996;46:102-111.
19 沈永才，袁佩娜．利用16S rRNA的PCR法鉴定双歧杆菌．中华微生物学和免疫学杂志 2001；21：578-579．
20 Langendijk PS, Schut F, Jansen GJ,et al. Quantitative fluorescence in situ hybridization of Bifidobacterium spp. with genus-specific 16S rRNA-targeted probes and its application in fecal samples. Ap—pl Environ Microbiol 1995;61:3069-3075.
21 Harmsen HJ, Raangs GC, He T, et al. Extensive set of 16S rRNA-based probes for detection of bacteria in human feces. Appl Environ Microbiol 2002;68:2982-2990.
22 Steidler L. In situ delivery of cytokines by genetically engineered Lactococcus lactis. Antonie Van Leeuwenhoek 2002;82:323-331.
23 魏曦．主编．正常菌群与健康．上海：上海科技出版社 1985：25-94．
2、张达容 微生态学在医学领域中的应用[J] 中国微生态学杂志 2002，2(14)3-4，
3、Steidler L, In situ delivery of cytokines by genetically engineered Lactococc us lactis [J] Antonie Van Leeu wenhoek, 2002,82(14):323-231
4. Gorbach SL, Plaut AG, Nahas L, et al, Studies of intestinal microflora. Gastroenterology, 1967, 53:856
5．潘令嘉等 正常国人小肠腔菌群及膜菌群的研究[J]中华内科杂志 1995，35(10)；686
6．潘令嘉等 小肠内容物腔菌群及膜菌群的研究[J]中国微生态学杂志1996，8(6)1-8，
7．熊得鑫．厌氧菌得分离和鉴定方法．南昌：江西科学技术出版社，1986．163-173
8．熊得鑫．使用悉生大鼠建立肠道，膜菌群的方法．中国微生态杂志，1993，5(2)：47
9 Molin G, Jeppsson B, Johansson ML,et al. Numerical taxonomyof different lactobacillus spp associated with healthy and diseasedmucosa of the human intestines. J Appl Bacteriol,1993,74 (3):314
10 Johansson ML, Molin G, Jeppsson B, et al. Administration of different lactobacillus strains in fermented oatmeal soup: in vivo colonization of human intestinal mucosa and effect on the indige2nous flora. Appl Environ Microbiol,1993,59 (1): 15
1 康白．人类胃肠道微生态学．见：康白．主编．微生态学．第1版．大连：大连出版社 1998：200-221．
2 康白．正常微生物群的生理代谢．见：康白．主编．微生态学．第1版．大连：大连出版社 1998：82-93．
3 康白．正常微生物群与其病变的相互关系．见：康白．主编．微生态学．第1版．大连：大连出版社 1998：64-80．
4 迟明，李颜．微生态制剂在儿童肠道疾病的应用进展．中国微生态学杂志2006；18：80．
5 Lee MC, Lin LH, Hung KL, et al. Oral bacterial therapy promotes recovery from acute diarrhea in children. Acta Paediatr Taiwan 2001;42:301-305.
6 黄柳革．金双歧、思密达联合治疗小儿急性腹泻病的疗效观察．中国微生态学杂志 2005；17：39-40．
7 董文岚．微生态制剂(金双歧)治疗婴幼儿腹泻60例临床观察．中国微生态学杂志 2005；17：40．
8 崔海宏，陈村龙，孙勇，等．溃疡性结肠炎患者肠黏膜菌群改变及抗体反应．中国微生态学杂志 2003；15：151-152．
9 Guslandi M, Mezzi G, Sorghi M, et al. Saccharomyces boulardii in maintenance treatment of Crohn's disease. Dig Dis Sci 2000;45:1462-1464.
10 Dunne C. Adaptation of bacteria to the intestinal niche: probiotics and gut disorder. Inflamm Bowel Dis 2001;7:136-145.
11 Bergogne-Berezin E. Treatment and prevention of antibiotic associ—ated diarrhea. Int J Antimicrob Agents 2000;16:521-526.
12 Olah A, Belagyi T, Issekutz A, et al. Randomized clinical trial of specific lactobacillus and fibre supplement to early enteral nutrition in patients with acute pancreatitis. Br J Surg 2002;89:1103-1107.
13 蒋虹，胡宏．双歧杆菌表面分子诱导大肠癌细胞分化的研究．中华微生物学和免疫学杂志 2001；21：186-190．
14 王立生，潘令嘉，陈村龙，等．双歧杆菌对裸鼠移植大肠癌增殖与凋亡的原位研究．世界华人消化杂志 2000；8：429-431．
15 Challa A, Rao DR, Chawan CB, et al. Bifidobacterium longum and lactulose suppress azoxymethane-indueed colonic aberrant crypt fo—ci in rats. Carcinogenesis 1997;18:517-521.
16 杨景云，杨继新．中医药微生态学的研究现状．中国微生态学杂志2003；15：1-3．
17 刘佳文，吕红艳．16SrRNA基因序列在分枝杆菌分类鉴定的研究进展．国外医学临床生物化学与检验学分册 2001；22：265-268．
18 Leblond-Bourget N, Philippe H, Mangin I, et al. 16S rRNA and 16S to 23S internal transcribed spacer sequence analyses reveal inter- and intraspecific Bifidobacterium phylogeny. Int J Syst Bacteriol 1996;46:102-111.
19 沈永才，袁佩娜．利用16S rRNA的PCR法鉴定双歧杆菌．中华微生物学和免疫学杂志 2001；21：578-579．
20 Langendijk PS, Schut F, Jansen GJ,et al. Quantitative fluorescence in situ hybridization of Bifidobacterium spp. with genus-specific 16S rRNA-targeted probes and its application in fecal samples. Ap—pl Environ Microbiol 1995;61:3069-3075.
21 Harmsen HJ, Raangs GC, He T, et al. Extensive set of 16S rRNA-based probes for detection of bacteria in human feces. Appl Environ Microbiol 2002;68:2982-2990.
22 Steidler L. In situ delivery of cytokines by genetically engineered Lactococcus lactis. Antonie Van Leeuwenhoek 2002;82:323-331.
23 魏曦．主编．正常菌群与健康．上海：上海科技出版社 1985：25-94．