【作者】 王慧；

【导师】 陈村龙； 张建中；

【作者基本信息】 南方医科大学 ， 内科学， 2009， 硕士

【摘要】 目的和意义研究目前国内外普遍关注临床上大量采用的幽门螺杆菌根除治疗方案在肠道微生态平衡打破方面的作用及其对肠道微生态的影响。本研究通过16S rDNA基因序列分析观察质子泵抑制剂（proton pump inhibitor,PPI）、阿莫西林、克拉霉素组成的三联疗法根除治疗幽门螺杆菌（Helicobacter pylori,H.pylori）感染对肠道菌群的影响。通过研究三联疗法根除H.pylori感染对肠道微生态平衡打破方面的作用及其对肠道微生态的影响,对于指导临床用药,研究新的抗H.pylori感染治疗方法以及减少药物副作用等具有潜在意义;同时为构建环境和临床标本中微生物的快速测定基因芯片提供研究基础。方法1.研究对象20-25名志愿者被告知将接受一次¹³C尿素呼气试验,该试验对机体无任何损害,对H.pylori感染诊断的敏感性和特异性高于90%。这些志愿者均填写知情同意书并接受一次问卷调查,合格的志愿者符合下列标准:¹³C呼气试验阳性,且近5周内未使用任何制酸剂、益生菌、抗生素或和抗微生物制剂,消化系统无其它器质性疾病,消化道未进行过手术活动,无酗酒史,无糖尿病及影响肠道菌群的其它问题。最后共有12名志愿者符合情况,包括8名男性和4名女性,平均年龄为34岁（年龄范围为23-58岁）。所有的志愿者都填写了知情同意书。被¹³C呼气试验证明为H.pylori感染的12名志愿者在浙江大学医学院第一附属医院口服给予为期7天的标准三联疗法（阿莫西林1000mg,2次/天,饭后服;克拉霉素500mg,2次/天,饭后服;雷贝拉唑20mg,2次/天,饭前服）,对治疗期间出现的药物副作用详加记录。这些志愿者治疗期间的饮食习惯与平常一样,治疗期间所有志愿者不再服用任何制酸剂、益生菌、抗生素,治疗结束后4周复查¹³C呼气试验评价是否根除成功。2.标本收集每个志愿者在接受治疗前一天（0天）和一周疗法结束后一天（第8天）各采集粪便10克。采集的粪便与10ml生理盐水混匀,分装10支（每支1ml）至1.5ml液氮冻存管于液氮中保存。标本采集完全后,运送到中国疾病预防控制中心传染病控制所诊断室的实验室,所有粪便标本-72℃保存。3.细菌DNA提取每份标本取0.5ml（约相当于0.5g粪便）用GENMED粪便细菌DNA分离试剂盒自粪便标本中提取细菌总DNA。提取的核酸用紫外分光光度计测定其浓度。4.PCR扩增及其产物纯化利用2个通用引物扩增细菌16S rRNA基因的全长,PCR扩增选用细菌通用引物序列为:8F（5’-AGAGTTTGATCATGGCTCAG-3’）和1492R（5’-ACGGTTACCTTGTTACGACTT-3’）,该引物对绝大数细菌16S rDNA基因序列具有特异性。PCR反应混合液包括0.5μl EXTaq酶(2.5u ml^-1,T a K a Ra生物,中国大连),5μl 10×EXTaq Buffer(plusMg²⁺),4μl dNTP mixture（各2.5MM）,上下游引物（10μmol/L）各0.5μl,8-Methoxypsoralen（8-MOP）0.5μl（2.5mg/ml）,去离子水定容至49μl,将未加模板的混合液在室温下用紫外线照射后再加1μl模板;PCR反应条件:95℃预变性5min;95℃变性30s、52℃退火1min、72℃延伸1.5min,共25个循环;72℃最后延伸5min,阴性对照用去离子水代替模板,扩增反应在My Cycler^TM thermal cycler上进行。PCR产物用经GelRed^TM染色的1%琼脂糖凝胶电泳（缓冲液为1×TAE buffer）鉴定,图像用GelDoc凝胶成像仪观察,1.5kb DNA条带自凝胶上切下,PCR产物纯化按Qiaquick PCR gel extraction kit说明书操作步骤进行。5.16S rDNA克隆文库的建立和序列分析经纯化的PCR产物与pGEM-T easy载体连接,然后按操作说明转入大肠杆菌DH5a感受态细胞,重组的克隆子在LB琼脂平皿（包括100mg/ml的氨苄青霉素,20mg/ml的X-Gal和24mg/ml的IPTG）上生长,每份标本随机挑选100个白色克隆子传代于含100mg/ml氨苄青霉素的LB平皿上,37℃培养过夜后,将克隆子送公司测序。短期4℃保存连接后转化克隆平皿,若测序中需补充克隆时,进行补充挑选。用CHIMERA-CHECK程序检测序列中可能出现的嵌合体,每个16S rDNA序列（大约1500bp）同时用GenBank的BLAST和RDP数据库进行比对,若某个序列在两个数据库中同时为一类则该序列归为此类,若某个序列在两个数据库不匹配则按RDP数据库中最相似的进行归类。6、统计分析治疗前和治疗后细菌特征的改变如培养条件、致病性用卡方检验进行统计分析,p＜0.05被认为差异具有统计学意义。统计软件使用SPSS11.0。结果1.治疗期间出现的副反应志愿者平常为成形固体便或稍硬便。治疗期间未发现严重的腹泻,最常见的副作用是轻微腹泻、口苦和轻微腹痛。在三联疗法治疗期间,有10个人出现腹泻,3个人出现口苦,2个人轻微腹痛,1个人便秘。这些志愿者中有人只出现上述副反应中的一种,有人同时出现上述副反应的两种或两种以上。治疗期间未有人中断治疗。2.根据16S rDNA克隆文库分析肠道菌群随机挑选的2400个克隆子的经RDP和GenBank数据库比对共检测到39种细菌种属。治疗前粪便优势菌属有Bacteroides spp.,Lachnospiraceae Incertae Sedis,Peptostreptococcaceae Incertae Sedis,Ruminococcus spp.,Parabacteroides spp.,Faecalibacterium spp.,Escherichia coli,Roseburia spp.,Alistipes spp.,andStreptococcus spp.。治疗结束后粪便中主要菌属有Bacteroides spp.,Shigella spp.,Lachnospiraceae Incertae Sedis,Parabacteroides spp.,Escherichia coli.,Klebsiellapeneumoniae,Faecalibacterium spp.,Citrobacter spp.,PeptostreptococcaceaeIncertae Sedis and Ruminococcus spp。本研究中未检测到Bifidobacterium spp.,Lactobacillus spp.,and yeast。与治疗前相比,治疗后专性厌氧菌明显下降（χ²=51.040,p=0.000）,而兼性厌氧菌（χ²=190.678,p=0.000）及需氧菌（χ²=18.182,p=0.000）则明显增加。与治疗前相比,治疗后正常菌群的数量明显受到抑制（χ²=83.619,p=0.000）,而机会致病菌的数量如E coli and K.pneumoniae明显增加（χ²=124.101,p=0.000）,致病菌如Shigella于治疗后明显增加（χ²=468.127,p=0.000）。志愿者F于治疗后出现Campylobacter jejuni致病菌,治疗前未检测到该致病菌。结论根除H.pylori感染的三联疗法可以对12名中国志愿者的肠道菌群产生负面影响,这主要是因为肠道正常菌群的生态平衡被打破从而导致机会致病菌和致病菌过度生长使肠道抗定植力减弱。在临床实践中进行以抗生素为基础的根除H.pylori感染治疗时应该考虑到这方面的问题。本研究成功构建了根除HP治疗前后肠道菌群的16S rRNA基因克隆文库。通过16S rRNA序列分析技术,我们能够直接使用临床标本,不需要获得纯培养的细菌,能够快速检测和鉴定不能培养的细菌,鉴定使用常规方法难以鉴定的细菌和发现新的病原菌。16S rRNA序列分析技术可用于临床和环境中微生物的检测及鉴定,并能够较好的反映菌群的数量及多样性。我们的研究没有进一步证据证明抗生素根除H.pylori感染后对肠道微生态的长期影响,但是已有研究显示根除H.pulori感染治疗后35天左右受到抑制的肠道菌群回复正常。未来的研究应该关注目前的治疗方法是否将导致肠道菌群抗定植模式的长期改变。更多还原

【Abstract】 ObjictiveThe aim of this study was to investigated ecological disturbance of intestinal microflora caused by H.pylori triple therapy and its impact on gut microflora.The present study was to use sequence analysis of 16S rDNA genes to investigated changes in the intestinal microflora resulting from administration of an eradication regimen for Helicobacter pylori infection using proton pump inhibitor （PPI）,amoxycilin and clarithromycin.By study the triple therapy in eradication treatment for H.pylori infection on the impact of intestinal microecology,it is important for guiding clinical administer、study new anti- H.pylori infection therapy regimen and reduce side effects of drug.To provide research evidence for establish a rapid diagnostic method based on gene chip,which would apply to detect microorganisms in environmental and clinical specimens.Methods1.Study participantsFor about 20 to 25 volunteers were infomed to carry out ¹³C-urea breath test,it qualitatively detects active infection with sensitivity and specificity of more than 90 percent and the test without any damage to the body.The volunteers were signed a written informed consent form and these volunteers were recived a questionnaire survey.Eligible volunteers were selected based on the following criteria:patients with ¹³C-urea breath test positive and took no any antiacids,probiotics,antibiotics and/or antimicrobial agents 5 weeks prior to the study,had no other organic diseases of the digestive system and no previous surgical procedures on the digestive tract,no alcoholism,no diabetes or any other problem that could affect the intestinal flora. Finally,there were 12 eligible volunteers involved in the study,including 8 men and 4 women,with a mean age of 34 years（range 23-58 years）.The informed consent was obtained from all volunteers.Twelve volunteers with H.pylori infection was verified by a positive urease test were administered orally,for 7-d standard triple therapy（amoxycilin 1000mg bid, take after meals;clarithromycin 500mg bid,take after meals;rabeprozole 20mg bid,take before meals ）at the First Affiliated Hospital of ZheJiang Unicersity School of Medicine,HangZhou,China.The side effects of the drug were observed and recorded throughout the treatment period.These individuals were lived on a Chinese diet as usually they does during the treatment.All the volunteers were not took any other antiacids,probiotics,antibiotics agents during the treatment and underwent a ¹³C-breath test to assess the eradication of H.pylori infection four weeks after treatment.2.Sample collectionAn accurately weighed 10g portion of stool samples were collected from each volunteer before（day 0）,1 day after（day 8） take medicine.Each fecal sample was mixed well and diluted in 10ml of 0.9%sodium chloride and to reserve immediately in 1.5ml frozen tube（1ml per tube,10 tube total per fecal sample ）and frozen in liquid nitrogen.After the entire specimen collection,all samples were transported to the laboratory of Chief of Department of diagnosis,National Institute for Communicable Disease Control and Prevention（NICDC）,China CDC.All fecal samples were frozen at -72℃.3.Bacterial DNA extractionThe total DNA from each diluted fecal sample（0.5ml,about 0.5g stool） was extracted by GENMED Stool Bacterial DNA Isolation Kit（GENMED SCIENTIFICS INC.U.S.A） as described by the manufacturer’s instruction.The concentration of nucleic acids was detected by using UV spectrophotometer（Eppendorf AG,Germany） at 260nm.4.Amplification of bacterial 16S rDNA and purification of productsThe near-full sequence of bacterial 16S rDNA were amplified with two-universal primers 8F（5’AGAGTTTGATCCTGGCTCAG 3’,Designed for most eubactria,E. coli positions 8-27） and 1492R（5’ACGGTTACCTTGTTACGACTT 3’,Designed for enterics and most eubactria,E.coli positions 1492-1512）,which are specific for universally conserved bacterial 16S rDNA gene sequences.The PCR reaction mixture,containing 0.5μl of 2.5u ml^-1 EX Taq polymerase（TaKaRa Biotechnology （Dalian,China） Co.,Ltd.）,5μl of 10×EXTaq Buffer(plusMg²⁺),4μl of dNTP mixture（2.5MM each）,0.5μl of 10μmol L^-1 each primer,0.5μl of 2.5mg ml^-1 8-methoxypsoralen（Sigma Aldrich Chemie GmbH,Steinheim,Germany:）,was made up to 49μl with DNA-free water.A DNA suspension was added last in a 1μl volume after irradiation of the PCR reaction mixture with UV light:The tubes containing the PCR mixture were irradiated from above at a distance of 1 cm with long-wave （320 nm） UV at room temperature.After an initial denaturation step of 95℃for 5min,samples were subjected to 25 cycles of denaturation at 95℃for 30s,annealing at 52℃for 60s,and extention at 72℃for 90s,with an additional step extentional at 72℃for 10min.Amplification was carried out by using a My Cycler^TM thermal cycler（Bio-Rad CO.,U.S.A）.PCR products were confirmed using 1%（wt./vol.） agarose gel electrophoresis in 1×TAE buffer（40mM Tris,20mM acetic acid,1mM EDTA,pH8.3）after GelRed^TM staining（Biotium,inc.,Hayward,U.S.A）and images captured by using a GelDoc image（Bio-Rad CO.,U.S.A）.About 1.5kb size of DNA band was observed from all the 24 PCR amplicons of fecal samples.The 1.5kb DNA band was excised from the gel and the DNA was purified with a QIAGEN PCR gel Extraction Kit according to the manufacturer’s protocals.5.Construction of 16S rDNA clone libraries and sequence analysis The purified PCR products were ligated into pGEM-T easy vector （Promega,Madison,USA） and then transformed into E.coli DH5a competent cells as described by the manufacturer’s instruction（TIANGEN BIOTEC CO.,LTD,Beijing, China）.Recombinant colonies were grow on Luria-Bertani（LB）agar plates which contained 100mg ml^-1 Ampicillin,20mg ml^-1 X-Gal and 24mg ml^-1IPTG（TIANGEN BIOTEC CO.,LTD,Beijing,China）.One hundred white colonies were randomly picked from each sample and growm in LB plates containing 100mg ml^-1 Ampicillin. Sequening of colonies carried out by the Tsingke Biotec CO.,LTD,Beijing,China. The sequence were examzed for possible chimeric artifacts using the programs CHIMERA-CHECK.Each 16S rDNA sequence（mostly around 1500 base pairs） was analyzed by BLAST search against GenBank（http://blast.ncbi.nlm. nih.gov/Blast.cgi） and by using the Ribosomal Database ProjectⅡ（RDP,http:// rdp.cme.msu.edu/）.If a sequence was well-matched within two datebases,the clone was placed into the corresponding taxon.If a sequence was not macthed each other within two datebases,it was determined which sequence in RDP were the most similar.6、Statistical analysisStatistical analysis was performed using Chi-square test,to compare pre-treatment and post-treatment changes in the characteristics of the kinds of bacterium.Only p＜0.05 were considers as significant differences were considered （use SPSS 11.0 for statistical analysis）.Results1.Adverse events of the volunteers during the treatmentThe habits of defecation of the volunteers in the usually condition is solid and rather hard.In this study severe diarrhoea was never observed during the treatment.The most common adverse events were mild diarrhoea,taste disturbance and mild abdominal pain.During the week of triple-drug regimen,diarrhea and loose stools were reported by 10 volunteers,taste disturbance were found in 3 volunteers,2 volunteers experienced mild abdominal pain and 1 volunteer experienced constipation,in no case leading to discontinuation of treatment.2.Aanlysis of the intestinal microorganism profiles according 16S rDNA librariesTotally,39 different kinds of genera from a total of 2400 clones were randomly selected from fecal samples were identified based on 16S rDNA sequence homology search through GenBank and RDP database.The predominance genera of the facal samples before treatment were found to be Bacteroides spp.,Lachnospiraceae Incertae Sedis,Peptostreptococcaceae Incertae Sedis,Ruminococcus spp.,Parabacteroides spp.,Faecalibacterium spp.,Escherichia coli,Roseburia spp.,Alistipes spp.,and Streptococcus spp.that included more number than other genera.The major genera of the facal samples after completion of the therapy（on 8 days） were found to be Bacteroides spp.,Shigella spp., Lachnospiraceae Incertae Sedis,Parabacteroides spp.,Escherichia coli.,Klebsiella peneumoniae,Faecalibacterium spp.,Citrobacter spp.,Peptostreptococcaceae Incertae Sedis and Ruminococcus spp.that included more number than other genera. In this study,Bifidobacterium spp.,Lactobacillus spp.,and yeast were not detected.The study shows immediately after treatment,the number of obligate anaerobe （χ²=51.040,p=0.000 ） are decreased significantly while the facultative anaerobe （χ²=190.678,p=0.000 ） and aerobe（χ²=18.182,p=0.000 ） are increased than before treament.As compared with before-therapy,the number of normal flora （χ²=83.619,p=0.000 ） are strongly suppressed and the number of opportunistic pathogen（χ²=124.101,p=0.000 ） such as Escherichia coli.and Klebsiella spp.are increased significantly after-therapy,and pathogenic bacterium（χ²=468.127, p=0.000 ） such as Shigella are increased significantly after treatment.The pathogenic bacterium Campylobacter was detected on one individual（volunteer F） only after treatment,which was not detected on any other individual before treatment.ConclusionsFrom our observation it can be concluded that there has been a negative effects on the normal intestinal flora in 12 Chinese volunteers because of marked ecological disturbances in the normal gut microflora that may lead to overgrowth of opportunistic pathogen and pathogenic bacterium and may decreased colonization resistence which associated with administered antimicrobial agents during the triple-drug regimen eradication treatment for H.pylori infection.This aspect should be taken into consideration when an antimicrobially based treatment for H.pylori infection in the clinical practice.In recent year,some study have indicated probiotics and prebiotics in H.pylori eradication has been used supplementally for the protection of ecologic balance of gut microflora while releasing the severity of the antimicrobial agents adverse effects and increase patient compliance,may be this is a way to improve the disturbance of intestinal microflora during treatment with antimicrobial angents.Our stduty have no further evidence that whether there is a long-term impact on the gut microflora ecosystem after eradication H.pylori with antibiotics,but one study have indicated that the suppression of nomal intestinal flora had almost reversed 35 days after the initiation of treatment for H.pylori infection.Future studies should pay attention to whether the present H.pylori triple therapy will result in persistent changes in colonization resistence pattern of intestinal microflora.更多还原