益生菌抑制溃疡性结肠炎癌变的相关机制及肠道菌群差异分析

发布时间：2018-04-16 20:21

本文选题：益生菌 + 溃疡性结肠炎癌变　；参考：《北京协和医学院》2017年博士论文

【摘要】：第一部分益生菌对溃疡性结肠炎癌变的抑制作用及肠道菌群分析目的:分析益生菌VSL#3、布拉氏酵母菌干预对AOM/DSS诱导的溃疡性结肠炎癌变模型小鼠成瘤的影响,同时分析小鼠粪便和黏膜菌群的变化,探讨肠道菌群变化在UC癌变中的作用。方法:C57BL/6小鼠随机分为7组。第1-6组建立溃疡性结肠炎癌变模型,第1周第1天予腹腔注射12.5mg/kg AOM,1周后开始连续5天予含2.5%DSS的饮用水,随后更换成普通饮用水。其中第1-5组为不同干预组,即从注射AOM当日起分别予 5-ASA(75mg/kg/d)、布拉氏酵母菌(5×107CFU/d)、VSL#3(1.5×l09CFU/d)、5-ASA联合布拉氏酵母菌、5-ASA联合VSL#3灌胃处理(5d/周)。第6组为模型对照组,不予灌胃干预。第7组为空白对照组,不予建模及灌胃干预。第12周末处死小鼠,统计比较各组瘤负荷。收集试验前后小鼠粪便及试验后肠黏膜标本,通过16s rDNA扩增子测序方法分析各组肠道菌群差异。结果:1.各组小鼠成瘤率:干预组63-85%,模型对照组100%。2.各组小鼠瘤负荷:5-ASA 组(0.43±0.14cm)、布拉氏酵母菌组(0.20±0.07cm)、VSL#3 组(0.25±0.07cm)、5-ASA+布拉氏酵母菌组(0.53±0.1lcm)、5-ASA+VSL#3 组(0.46±0.11cm)瘤负荷较模型对照组(0.97±0.19cm)均显著下降,差异有统计学意义(p=0.0269,p=0.0006,p=0.0009,p=0.0486,p=0.0261)。3.粪便菌群分析显示:(1)模型对照组较空白对照组乳杆菌属减少,颤杆菌属、Lachnoclostridium增加。(2)益生菌干预后,芽孢杆菌属、乳球菌属、乳杆菌属增加,颤杆菌属、Lachnoclostridium、拟杆菌属、假单胞菌属减少。4.肠道黏膜菌群分析显示:(1)模型对照组较空白对照组瘤胃球菌科014菌属、双歧杆菌属减少,拟普雷沃菌属增加。(2)较模型对照组相比,布拉氏酵母菌组毛螺旋菌科NK4A136菌属、Lachnoclostridium增加;VSL#3组双歧杆菌属增加。结论:l.VSL#3和布拉氏酵母菌能够显著降低UC癌变小鼠的成瘤率和瘤负荷。2.UC癌变小鼠肠道菌群紊乱,黏膜和粪便菌群均表现为有益菌属减少、有害菌属增加。3.补充益生菌有益于UC癌变小鼠粪便和黏膜菌群实现再平衡。4.双歧杆菌属容易在肠黏膜定植,芽孢杆菌属和乳球菌属容易在粪便定植。第二部分炎症因子及布拉氏酵母菌对Wnt信号通路的影响及机制研究目的:探讨炎症因子IL-6、TNF-α及布拉氏酵母菌对Wnt信号通路的影响及作用机制。方法:选用正常肠黏膜上皮细胞CCC-HIE-2、结肠癌细胞Caco-2和HCT-116,分别予IL-6和TNF-α刺激,采用双荧光素酶报告基因实验检测Wnt信号通路活性;给予NF-(?)B信号通路特异性抑制剂PDTC,观察PDTC对IL-6和TNF-α调节Wnt信号通路的影响;将布拉氏酵母菌与细胞共培养,观察对IL-6和TNF-α调节Wnt信号通路的影响。结果:1.不同浓度IL-6和TNF-α刺激CCC-HIE-2、Caco-2、HCT-116均能使相对荧光素酶活性升高,上调Wnt信号通路。IL-6和TNF-α刺激细胞的最佳浓度及时间均为 50ng/ml,6h。2.PDTC 可以抑制 IL-6 和 TNF-α 对 CCC-HIE-2 细胞(1.044+0.006 vs 0.895±0.003 p=0.0032,1.126±0.006 vs l.062±0.014/p=0.0006)、Caco-2 细胞(8.859±0.050 vs 7.922±0.075 p=0.0046,3.951±0.006 vs 3.550±0.022 p=0.0016)和 HCT-116 细胞(2.728±0.053 vs2.430+0.022p=0.0008,4.044±0.022 vs 3.629±0.025p=0.0033)的上调相对荧光素酶活性作用。3.布拉氏酵母菌可以抑制 IL-6 和 TNF-α 对 CCC-HIE-2 细胞(1.239±0.015 vs 0.956±0.001 p=0.0014,1.022±0.014 vs 0.780±0.026 p=0.0074)、Caco-2 细胞(9.731±0.075 vs 8.479±0.099 p=0.0049,9.187±0.044vs7.691±0.055p=0.0011)和 HCT-116 细胞(4.116±0.004 vs3.734±0.045p=0.0068,4.489±0.010vs 3.858±0.021p=0.0007)的上调相对荧光素酶活性作用。结论:1.在 CCC-HIE-2、Caco-2、HCT-116 细胞系,TNF-α 和 1L-6 能够通过 NF-(?)B通路上调Wnt信号通路。2.布拉氏酵母菌与CCC-HIE-2、Caco-2、HCT-116细胞共培养,能够抑制TNF-α和IL-6对Wnt信号通路的上调。第三部分不同病变范围溃疡性结肠炎患者肠道菌群分析目的:探讨不同病变范围溃疡性结肠炎患者肠道菌群特点。方法:收集10例健康体检个体粪便标本,35例UC患者(直肠型7例,左半结肠型9例,广泛结肠型19例)粪便及左半、右半肠黏膜标本,通过16srDNA扩增子测序方法分析肠道菌群差异。结果:1.粪便菌群分析显示:(1)广泛结肠型UC患者较健康个体菌群多样性显著减少。(2)较健康个体相比,UC患者Subdoligranulum、瘤胃球菌属-2、Fusicatenibacter、柔嫩梭菌属、假丁酸弧菌属、Alistipes减少;埃希氏菌属、Peptoclostridium属及韦荣氏菌属增加。(3)直肠型UC较左半结肠型UC患者粪便菌群假丁酸弧菌属增加,较广泛结肠型UC患者毛螺旋菌属增加、普雷沃氏菌属-9减少。2.黏膜菌群分析表明:(1)较直肠型患者相比,左半结肠型和广泛结肠型患者乳球菌属、Geobacillus、链球菌属、明串珠菌属减少。(2)较广泛结肠型患者相比,直肠型和左半结肠型患者拟杆菌属减少。(3)所有类型UC患者左半与右半黏膜菌群生物多样性及菌属组成无统计学差异。(4)UC患者肠道黏膜较粪便菌群生物多样性增多,菌属组成存在差异。结论:1.UC患者粪便菌群生物多样性降低,有益菌属减少,有害菌属增加。2.随着病变范围增大,患者粪便和肠黏膜菌群均表现为有益菌属减少,有害菌属增加。3.患者黏膜菌群的改变呈全结肠分布改变特点,不局限于病变部位黏膜。4.患者粪便和黏膜菌群生物多样性及菌属组成存在差异。目的:探讨胃神经内分泌肿瘤(Gastric Neuroendocrine Neoplasms,GNENs)的临床特征,以提高临床医师对该病的认识。方法:收集2005年10月至2015年10月北京协和医院收治的36例GNENs,对其人口学特征、主要临床表现、内镜下表现、病理特征及治疗情况进行回顾性分析。结果:1.36例患者平均年龄55.8岁,男女比例0.89:1。2.临床表现多种多样,腹痛的发生率高达42.9%。3.63.9%的病变分布于胃体部。内镜下病变可表现为息肉样隆起、溃疡性病变、黏膜凹陷。以息肉样隆起最为多见,其中73.9%为单发病变,平均直径小于l0mm。4.病理分级包括G1-G3级,以G1级居多(55.6%)。5.65.2%于胃体行活检的患者病理证实存在胃体黏膜萎缩或肠化。6.治疗方案主要包括内镜和手术根治。结论:1.GNENs缺乏特异性临床表现,病变以胃体分布居多,在内镜下多表现为息肉样隆起。2.对于合并萎缩性胃炎,尤其胃体萎缩的息肉样病变,应警惕GNENs的可能性。
[Abstract]:The first part of probiotics inhibit carcinogenesis of ulcerative colitis and intestinal microflora analysis objective: analysis of probiotics VSL#3 intervention ofsaccharomyces boulardii tumor effect on mice ulcerative colitis carcinogenesis model induced by AOM/DSS, and the analysis of mice feces and mucosal flora changes, to explore the changes of intestinal microflora in UC carcinogenesis. Methods C57BL/6 mice were randomly divided into 7 groups. The 1-6 group to establish ulcerative colitis carcinogenesis model, first weeks and first days by intraperitoneal injection of 12.5mg/kg AOM, 1 weeks after the start of 5 consecutive days to drinking water containing 2.5%DSS, then replace the ordinary drinking water. The 1-5 group is different from the intervention group, the injection of AOM since they were given 5-ASA (75mg/kg/d), Saccharomyces boulardii (5 * 107CFU/d), VSL#3 (1.5 * l09CFU/d), 5-ASA and 5-ASA combined with VSL#3 boulardii, gavage treatment (5d/ weeks). The sixth groups as model control group, not Stomach intervention. The seventh group was the control group, no modeling and stomach intervention. At the end of the twelfth mice were killed, were collected. Statistical tests before and after the tumor burden mice feces and test of intestinal mucosa specimens by 16S rDNA amplicon sequencing method to analyze the difference of intestinal flora. Results: 1. groups of mice: the intervention rate group 63-85%, model group 100%.2. mice tumor burden: group 5-ASA (0.43 + 0.14cm), Saccharomyces boulardii group (0.20 + 0.07cm), group VSL#3 (0.25 + 0.07cm), 5-ASA+ group of Saccharomyces boulardii (0.53 + 0.1lcm), group 5-ASA+VSL#3 (0.46 + 0.11cm) tumor burden compared with model control group (0.97 + 0.19cm) were significantly decreased, the difference was statistically significant (p=0.0269, p=0.0006, p=0.0009, p=0.0486, p=0.0261).3. fecal flora analysis showed that: (1) the model group compared with blank control group Lactobacillus decreased, Lachnoclostridium increased. AF bacillus, probiotics (2) After the intervention, bacillus, Lactococcus, Lactobacillus, Bacillus Lachnoclostridium, fibrillation, Bacteroides spp., Pseudomonas.4. reduce the intestinal mucosal flora analysis showed that: (1) model group compared with blank control group, 014 Ruminococcus genus, the genus Bifidobacterium reduced to Prevotella sp. is increased. (2) compared with the model group compared with the control group, Saccharomyces boulardii lachnospiraceae, NK4A136 sp., Lachnoclostridium increased in VSL#3 group; Bifidobacterium increased. Conclusion: l.VSL#3 and Saccharomyces boulardii can significantly reduce the rate of tumor and tumor load.2.UC cancer mice intestinal flora disorder UC cancer mice, mucosa and fecal bacteria the group showed good bacteria is reduced, harmful bacteria increased.3. probiotics is beneficial to UC cancer mice feces and mucosal flora to rebalance.4. Bifidobacterium easily in intestinal colonization, bacillus and Lactococcus. In the fecal colonization. Objective to study the effect of Wnt signal pathway and mechanism of the second part: To investigate the inflammatory factors and Saccharomyces boulardii inflammatory cytokine IL-6, TNF- alpha and Saccharomyces boulardii effect on Wnt signaling pathway and the mechanism. Methods: the CCC-HIE-2 of normal intestinal epithelial cells, Caco-2 and HCT-116 colon cancer cells were treated with IL-6 and TNF- alpha stimulation by Wnt signaling pathway activated luciferase reporter gene experiment; NF- (?) B pathway inhibitor PDTC, observe the PDTC Wnt signal pathway in regulating IL-6 and TNF- alpha will influence; Saccharomyces boulardii and cell co culture, observe the effect on IL-6 and TNF- alpha Wnt pathway results: 1. different concentrations of IL-6 and TNF- CCC-HIE-2 Caco-2, HCT-116 alpha stimulation, can make the relative luciferase activity increased, the optimum concentration of upregulation of Wnt signaling pathway of.IL-6 and TNF- stimulated cells And the time of 50ng/ml, IL-6 and 6h.2.PDTC can inhibit TNF- alpha on CCC-HIE-2 cells (1.044+0.006 vs 0.895 + 0.003 p=0.0032,1.126 + 0.006 vs l.062 + 0.014/p=0.0006), Caco-2 cells (8.859 + 0.050 vs 7.922 + 0.075 p=0.0046,3.951 + 0.006 vs 3.550 + 0.022 p=0.0016) and HCT-116 cells (2.728 + 0.053 vs2.430+0.022p=0.0008,4.044 + 0.022 vs 3.629 + 0.025p=0.0033 the relative luciferase activity) by.3. boulardii inhibits IL-6 and TNF- alpha on CCC-HIE-2 cells (1.239 + 0.015 vs 0.956 + 0.001 p=0.0014,1.022 + 0.014 vs 0.780 + 0.026 p=0.0074), Caco-2 cells (9.731 + 0.075 vs 8.479 + 0.099 p=0.0049,9.187 + 0.044vs7.691 + 0.055p=0.0011) and HCT-116 cells (4.116 + 0.004 + 0.045p=0.0068,4.489 + vs3.734 0.010vs 3.858 + 0.021p=0.0007) increased relative luciferase activity effect. On: 1. in CCC-HIE-2, Caco-2, HCT-116 cell lines, TNF- alpha and 1L-6 by NF- (?) B pathway. Wnt pathway.2. boulardii with CCC-HIE-2, Caco-2, HCT-116 cells were co cultured, can inhibit TNF- alpha and IL-6 on the up regulation of the Wnt signaling pathway. The third part is the same lesion in patients with ulcerative colitis the intestinal flora analysis objective: To investigate the different extent of disease in patients with ulcerative colitis intestinal flora characteristics. Methods: We collected 10 cases of healthy individual fecal samples from 35 UC patients (rectum type in 7 cases, 9 cases of left colon type extensive colonic type 19 cases) stool and left half, right through the intestinal mucosa specimens. 16srDNA amplicon sequencing method difference analysis of intestinal flora. Results: 1. fecal flora analysis showed that: (1) patients with extensive colon type UC compared with healthy individuals flora diversity decreased significantly. (2) compared with healthy individuals, UC patients with Subdoligranulum, rumen ball Genus of -2, Fusicatenibacter, and Clostridium, false butyrivibrio, Alistipes decreased; Escherichia coli, genus Peptoclostridium and genus Veillonella. (3) UC with left colon rectal UC patients fecal flora false butyrivibrio increased widely colonic type UC patients lachnospiraceae genus Poulet Was added, genus -9 reduced.2. mucosal flora analysis showed that: (1) a type of rectal patients with left colon and colon were widely Lactococcus, Geobacillus, Streptococcus, Leuconostoc. (2) compared with patients with extensive colitis, rectum and left colon in patients with type bacteriodes decreased. (3) all types of UC patients with left-sided and right-sided mucosal flora biodiversity and species composition had no significant difference. (4) UC with intestinal mucosa of patients with fecal flora biodiversity increased, species composition is different. Conclusion: 1.UC patients with fecal flora biology Reduced diversity, beneficial bacteria to reduce harmful bacteria is increased with.2., the lesion increased, and stool of patients with intestinal mucosa flora showed beneficial bacteria is reduced, harmful bacteria increased.3. mucosal flora of patients with changes in total colonic distribution characteristics, not limited to the lesion mucosa and mucosal.4. in patients with fecal bacteria group of biological diversity and species composition differences. Objective: To investigate the gastric neuroendocrine tumor (Gastric Neuroendocrine, Neoplasms, GNENs) of the clinical features, to improve the understanding of this disease. Methods: 36 cases of GNENs from October 2005 to October 2015 in Peking Union Medical College Hospital were, on the demographic characteristics, main clinical manifestations, endoscopic findings. The pathological characteristics and treatment were analyzed retrospectively. Results: 1.36 cases of patients with an average age of 55.8 years, the proportion of male and female 0.89:1.2. variety of clinical manifestations, the high incidence of abdominal pain As 42.9%.3.63.9% lesions distributed in the body of stomach. Endoscopic lesions showed polypoid bulge, ulcer lesions, mucosal polypoid bulge sag. In the most common, of which 73.9% were single lesions, the average diameter of less than l0mm.4. pathological grades including G1-G3 level, G1 level in the majority (55.6%) of patients with pathological.5.65.2% in the stomach body biopsies confirmed the presence of.6. treatment of gastric mucosa atrophy or intestinal metaplasia including radical endoscopy and surgery. Conclusion: 1.GNENs is lack of specific clinical manifestations. The lesion in the gastric body distribution mostly in endoscopic manifestations of polypoid bulge.2. for patients with atrophic gastritis, especially polypoid lesions of gastric atrophy. Should be alert to the possibility of GNENs.

【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R574.62

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