【作者】 王伟；

【导师】 夏冰； 赵秋；

【作者基本信息】 武汉大学 ， 内科学， 2015， 博士

【摘要】 目的：炎症性肠病(inflammatory bowel disease, IBD),主要包括溃疡性结肠炎(ulcerative colitis, UC)和克罗恩病(Crohn’s disease, CD),病因尚不明确,目前研究认为其发病机制由环境因素作用于遗传易感宿主引起持续异常的粘膜免疫反应失衡所致,其中肠道环境中的细菌起到关键作用。大量研究报道活动期IBD患者肠道菌群失调,然而,目前尚缺乏缓解期IBD患者微生态报道。因此,我们研究正常人群和IBD患者的粪便微生态特点,探讨肠道微生态与IBD发病的关系。方法：研究对象包括53例健康对照者,52例活动期克罗恩病患者,57例缓解期克罗恩病患者以及64例活动期溃疡性结肠炎患者和18例缓解期溃疡性结肠炎患者。收集纳入研究对象的粪便标本,提取细菌基因组DNA,基于变性梯度凝胶电泳(Denaturaing gradient gel electrophoresis, DGGE)分子指纹图谱技术及多元变量数据分析方法,探究活动期和缓解期炎症性肠病患者微生态的组成特点,基于实时定量聚合酶链反应验证PCR-DGGE结果的可靠性。结果：对收集的粪便样本采用PCR-DGGE指纹图谱方法进行细菌V3区16S rDNA条带分离,结果显示,IBD患者的肠道菌群结构与健康人群有显著差异。首先IBD患者的肠道菌群多样性显著下降。其次,这种差异表现为疾病活动期与缓解期均存在种特异性细菌丰度的改变；通过对DGGE图谱中的DNA条带位置校正分析后,共获得72条有效的band-classes,将band-classes转化为相对光强度进行校正分析后,发现14条差异性band-classes,通过分离、切胶测序,发现11个差异性band-classes由单一细菌构成。其中,band-class 2.44为马铃薯拟杆菌(Bacteroides vulgatus, B. vulgatus); band-classes4.06、4.37、4.57、4.76、4.99/5.99为厚壁菌门中的产丁酸盐细菌,序列比对分析后分别分配为Eubacterium eligens、Roseburia hominis A2-183、Eubacterium rectale、 Anaerosipes butyraticus、Faecalibacterium prausnitzii; band-classes 6.18、6.32经序列比对分析后均为大肠杆菌-志贺菌群(Escherichia coli-Shigella group); band-class 14.71为双歧杆菌属中的Bifidobacterium pseudocatenulatum、band-class16.72分配为厚壁菌门中的Collinsella aerofaciens。与健康对照相比,CD患者粪便中band-class 2.44的条带光强度(P。=1.58×10-5)和数量(P=0.004)显著下降,其光强度在缓解期中亦减少(Pc=4.65x 10-6),但其数量在活动期CD患者中无差异；Band-class 2.44光强度(Pc=0.03)和数量(P=0.001)在活动期UC患者也明显减少,缓解期UC患者条带光强度无统计学差异,但数量在缓解期UC患者中显著下降(P=0.015),活动期UC和缓解期UC患者间该细菌数量上无统计学差异。与健康对照相比,band-class 4.06(Eubacteria eligens, E. eligens)条带光强度(Pc=9.93×10-5)及数量(P=8.05×10-12)在活动期CD患者粪便中显著下降；在缓解期CD患者中,E. eligens条带光强度差异无统计学意义(Pc=0.07),但其数量显著下降(P=1.36×10-7)；活动期CD患者与缓解期CD患者相比,光强度和绝对数量均无统计差异,且E. eligens的数量变化与CD疾病活动度负相关。在活动期UC患者粪便中,E. eligens的光强度(Pc=0.3×10-3)和数量(P=1.47×10-9)亦显著下降,尽管缓解期E. eligens条带强度与健康对照相比差异无统计学意义,但其数量在缓解期UC患者中显著下降(P=0.006)。与健康对照相比,Band-class 4.37 (Eubacteria rectale, E. rectale)光强度(Pc=1.88×10-7)和数量(P=1.36×10-5)在活动期CD患者粪便群落中显著下降：在缓解期CD患者粪便群落DGGE图谱中,其光强度(Pc=2.76×10-7)和数量(P=0.018)亦处于较低水平；与活动期CD相比,缓解期CD患者粪便中E. rectale的数量(P=0.027)有上升趋势,但仍难达到正常水平。与健康对照比较,E. rectale条带光强度在活动期UC患者(Pc=5.06×10-13)和缓解期UC患者(Pc=0.017)中均显著下降,且活动期UC与缓解期UC患者差异无统计学意义；与健康对照相比,其数量在活动期UC患者中亦显著减少(P=5.97×105),但在缓解期UC患者中有上升趋势；活动期与缓解期之间无差异；此外,E.rectale数量变化水平与UC疾病活动度负相关(τ=-0.388,P=0.63 x 10-4)。与健康对照比较,Band-class 4.57 (Roseburia hominis A2-183,R. hominis A2-183)光强度在活动期CD(Pc=0.008)和缓解期CD患者(Pc=0.01)中均显著下降,且活动期与缓解期之间无统计学差异；其数量在活动期CD患者(P=1.55 x 10-16)和缓解期CD患者(P=1.34 x 10-14)亦显著下降,两者无统计学差异。在UC患者中,R. hominis A2-183光强度在活动期UC患者(Pc=1.24 x 10-5)和缓解期UC患者(Pc=0.0028)中均显著下降；其数量在活动期UC患者(P=1.04 x 10-15)和缓解期UC患者(P=9.70×10-8)中亦显著下降,且R. hominis数量变化与UC疾病活动度负相关(τ=-0.550,P=1.39 x 10-8)。Band-classes 4.99和5.99序列比对后均为普拉梭菌(Faecalibacterium prausnitzii, F. prausnitzii)。Band-class 4.99 F. prausnitzii光强度在活动期CD患者(Pc=1.56×10-5)和缓解期CD患者(Pc=0.04)中亦显著下降；但是,在UC患者中,band-class 4.99光强度与健康对照组的条带光强度值差异无统计学意义。Band-class 5.99光强度在活动期CD(Pc=0.047)和缓解期CD(Pc=0.048)患者中均下降,且活动期CD患者与缓解期CD患者条带强度无统计学差异；与健康对照相比,band-class 5.99光强度在UC患者粪便菌落中无统计学差异。F. prausnitzii的数量在活动期CD患者(P=4.61 x 10-10)和缓解期CD患者中(P=0.002)显著下降；与健康对照相比,在活动期UC患者中,F. prausnitzii的数量亦显著下降(P=0.003),但在缓解期UC中无统计学差异。F. prausnitzii数量变化水平与CD(τ=-0.502,P=4.50 x 10-6)和UC(τ=-0.429,P=9.43 x 10-6)严重程度均相关。序列比对分析发现band-classes6.18和6.32均为大肠杆菌-志贺菌群(Escherichia coli-Shigella group)。与健康对照相比,band-class 6.18光强度在活动期CD患者(Pc=0.0024)中显著升高,但在缓解期CD患者中其光强度差异无统计学意义,活动期和缓解期CD光强度无统计学差异；在活动期UC患者中,band-class 6.18检出率低于健康对照组(Pc=0.005),缓解期UC患者亦无统计学差异,活动期UC患者与缓解期UC患者条带光强度中位数无统计学差异。与健康对照组比较,band-class 6.32光强度在活动期UC患者(Pc=0.9×10-3)和缓解期UC患者(Pc=0.0068)中均升高,且活动期UC患者和缓解期UC患者band-class 6.32光强度无统计学差异,但CD患者中无统计学差异。与健康对照相比,Escherichia coli-Shigella group的数量在活动期CD患者粪便中(P=1.08×10-6)显著增加；在缓解期CD患者粪便样本中,其数量仍高于健康对照组(P=9.50×10-14)；其数量在活动期与缓解期CD中无统计学差异。在UC患者中,大肠杆菌群数量亦显著增加,尤其以活动期UC患者明显(P=1.06×10-7),缓解期UC患者粪便中大肠杆菌群数量较健康对照显著增加(P=0.03)；但与活动期患者相比,缓解期UC患者肠道中Escherichia coli-Shigella group的绝对数量有下降趋势,但两者无统计学差异。结论：IBD患者粪便菌群与健康对照者比较存在显著差异。活动期与缓解期CD和UC粪便菌群多样性均显著下降。活动期IBD患者粪便中存在产丁酸盐菌群失调,且失调的产丁酸盐细菌数量变化水平与IBD严重程度相关,表现为活动期CD患者中失调的E. eligens和F. prausnitzii数量变化水平与CD严重程度负相关,活动期UC患者中失调的R. hominis、F. prausnitzii、E. rectale变化水平与UC严重程度负相关；CD和UC患者具有相同的失调特征,均表现为F. prausnitzii数量减少且与疾病严重程度相关；缓解期IBD患者存在特征性菌群失调,表现为E. eligens, R. hominis失调,失调的产丁酸盐细菌在疾病缓解期可能难以恢复至正常水平。更多还原

【Abstract】 Objective Inflammatory bowel disease (IBD), mainly consist of ulcerative colitis (UC) and Crohn’s disease (CD), is chronic intestinal inflammatory disorders with unclear etiology. It is generally accepted that the pathogenesis of IBD involves an aberrant mucosal immune system respond towards intestinal commensal microflora antigens in genetically susceptible host. Substantial studies have reported the dysbiosis in active IBD, however, dysbiosis of microflora in quiescent IBD has not been proven. We therefore specifically sought to study the fecal microbiota of health controls and patients with IBD in different genetical background and diet characteristics from central china and explore the relationship between intestinal microbiota and pathogenesis of inflammatory bowel disease.Methods The study enrolled 53 cases of healthy controls (HC),52 cases of patients with active Crohn’s disease (ACD),57 cases of Crohn’s disease patients in remission (RCD),64 cases of patients with active ulcerative Colitis (AUC) and 18 cases of ulcerative Colitis patients in remission (RUC). We collected feces specimens and based on polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) fingerprint technology and multi-variate statistical method to analyze the composition and abundance of intestinal microbiota, and then verified the reliability of the results of the DGGE by real-time PCR.Results Overall, the gut flora of IBD patients presented a decrease on the level of diversity compared to the healthy individuals. Secondly, the abundance of species-specific bacteria significantly differed from healthy control in patients with IBD. We totally found 72 effective band-classes on DGGE fingerprint profiles using BioNumerics software, and 14 band-classes were discriminant significance in DGGE profiles after normalization with relative intensity, and 11 discriminant band-classes were aligned to different single species respectively. Of which, band-class 2.44 was aligned to Bacteroides vultagus (B. vultagus) belonging to Bacteroides genus. Band-classes 4.06,4.37,4.57,4.76,4.99/5.99 were assigned to Eubacterium eligens, Roseburia hominis A2-183, Eubacterium rectale, Anaerosipes butyraticus, Faecalibacterium prausnitzii respectively, of which belonging to Firmicutes phylum with butyrate-producing property. Both of band-class 6.18 and 6.32 were aligned to Eshcerichia coli-Shigella group. Band-class 14.71 and 16.72 were assigned to Bifidobacterium pseudocatenulatum and Collinsella aerofaciens respectively. Subsequently, we found that median intensity of band-class 2.44 and number of represented species decreased significantly in active CD compared to HC(Pc=1.58 x 10-5, P=0.004, respectively) and median intensity of band-class 2.44 also presented a significant decrease in RCD (Pc=4.65 x 10-6) compared to HC, but not in numbers in patients with RCD; we also found that median intensity of band-class 2.44 and numbers of its represented species decreased in active UC compared to HC (Pc=0.03, P=0.001, respectively), while we found count of this species decreased significantly in patients with RUC (P=0.015) compared to HC, but not relative intensity. Both of the relative intensity (Pc=9.93 x 10-5) of band-class 4.06 and amount (P=8.05 x 10-12) of represented species decreased significantly in ACD compared to healthy control in fecal specimens; although no significant decrease of relative intensity was observed in patients with RCD, number of its represented species decreased significantly in RCD (P=1.36 x 10-7) compared with HC, and a inverse correlation between the amount of E. eligens and disease activity (τ=-0.525, P=1.65 x 10-6); moreover, no significant difference was observed between RCD and ACD in numbers. The relative intensity (Pc=0.3 x 10-3) of band-class 4.06 and amount (P=1.47 x 10-9) of representd species E. eligens also decreased significantly in AUC compared to the HC, and we observed the count of E. eligens decreased significantly in RUC (P=0.006) compared with healthy control, but not relative intensity of E. eligens in patients with RUC. As for the band-class 4.37, relative intensity (Pc=1.88 x 10-7) and count (P=1.36 x 10-5) of represented species E. rectale decreased in ACD compare to HC, we also observed significant decrease of relative intensity (Pc=2.76 x 10-7) and number of represented species (P=0.018) in RCD compared to HC, and no significance of relative intensity and count were found between ACD and RCD; relative intensity of band-class 4.37 decreased significantly in both AUC (Pc= 5.06 x 10-13) and RUC (P=0.017) compared to healthy control, and the amount of represented species E. rectale significantly decreased in AUC (P=5.97 x 10-5) compared to HC, but not in RUC; and we found a inverse correlation between the amount of E. rectale and disease activity (τ=-0.388, P =0.63 x 10-4). We found relative intensity of band-class 4.57 decreased significantly in both ACD (Pc=0.008) and RCD (Pc=0.01) compared to HC, and the amount of represented species R. hominis A2-183 also decreased significantly in ACD (P=1.55 x 10-16) and RCD (P=1.34 x 10-14) respectively; moreover, relative intensity of band-class 4.57 displayed significant decrease in both AUC (Pc=1.24 x 10-5) and RUC (Pc=0.0028) compared to HC, and number of its represented species R. hominis also presented significant decrease in both AUC (P=1.04 x 10-15) and RUC (P=9.70 x 10-8) compared to HC; and a inverse correlation was found between the count of R. hominis and UC disease activity (τ=-0.550, P=1.39 x 10-8). Both of Band-class 4.99 and 5.99 were assigned to Faecalibacterium prausnitzii (F. prausnitzii), while relative intensity of band-class 4.99 presented significant decrease in both ACD (Pc=1.56 x 10-5) and RCD (Pc=0.04) compared to HC, but not in UC patients. We also found relative intensity of band-class 5.99 also decreased in both ACD (Pc=0.047) and RCD (Pc=0.048) comparetd to HC in feces, no significant difference was observed between ACD and RCD; however, no significant decrease in relative intensity of band-class 5.99 was observed in patents with UC. The count of F. prausnitzii significantly decreased in both ACD (P=4.61 x 10-10) and RCD (P=0.002) compared to HC; the numbers of F. prausnitzii significantly decreased in AUC (P=0.003) compare to HC, but no significant decrase was observed in RUC. The amounts of F. prausnitzii displayed inverse correlation with disease activity in both CD (t =-0.502, P=4.50 x 10-6) and UC (t=-0.429, P=9.43 x 10-6). Relative intensity of band-class6.18 presented a significantly increase in both active CD (Pc=0.0024) and active UC (Pc =0.005) compared to HC, while we didn’t find significant increasion in RCD and RUC. We found significant increase of relative intensity of band-class 6.32 in both AUC (Pc=0.9 x 10-3) and RUC (Pc=0.0068) compared to HC, but no significance was observed in patients with CD. Moreover, numbers of represented species of band-classes 6.18/6.32 Escherichia coli-Shigella group displayed dramatically increase in both ACD (P=1.08 x 10’6) and (P=9.50 x 10-14) compared to HC, significant increase also were observed in AUC (P=1.06 x 10-7) and RUC (P=0.03) in number, and no significance were observed in active and quiescent disease。Conclusion Significant structural shifts of fecal microbiota were observed in patients with IBD. These shifts were characterized by reduced diversity in both active and quiescent disease. Dysbiosis of butyrate-producing bacteria occurred in patients with IBD, and amounts of butyrate-producing bacteria presented inverse correlation with disease activity, characterizing by E. eligens in CD and R. hominis, F. prausnitzii and E. rectale in UC. Patients with CD and UC shared same dysbiosis characterized by inverse correlation between the amount of F. prausniitzii and disease activity. Distinct dysbiosis occurred in quiescent IBD characterized by decreased amount of E. eligens in CD and R. hominis in UC, and this dysbiosis could not be restored during disease in remission.更多还原