摘要
目的分析粪菌移植(Fecal microbiota transplantation,FMT)对葡聚糖硫酸钠(DSS)所致溃疡性结肠炎(UC)小鼠的治疗效果及免疫学机制。方法60只C57BL/6J小鼠,其中选取10只小鼠收集粪便(不参与后续实验),其余50只小鼠采用随机数字表法随机分为空白组(n=10)、模型组(n=20)、研究组(n=20)。空白组自分组首日起自由饮用灭菌蒸馏水,模型组和研究组自分组首日起自由饮用2%葡聚糖硫酸钠(DSS)溶液,诱导形成溃疡性结肠炎小鼠模型。小鼠模型建立成功后,模型组予以生理盐水灌肠,研究组予以正常小鼠粪便混悬液生理盐水混合物灌肠,每3天灌肠1次,共2次。待第2次灌肠完成后的第2天处死所有小鼠,采集血清样本。ELISA法检测血清中肿瘤坏死因子α(TNF-α)、白介素-6(IL-6)、白介素-35(IL-35)、白介素-22(IL-22)、白介素-17(IL-17)与可溶性Fas等细胞因子水平;HE染色观察结肠组织病理形态表现;光冈氏肠内细菌群分析法定量检测肠道菌群值及双歧杆菌与肠杆菌的数量比值(B/E值)。结果ELISA检测结果显示,与空白组比较,模型组和研究组小鼠TNF-α、IL-6、IL-17与IL-22水平上升,IL-35和可溶性Fas水平降低(P<0.05);与模型组比较,研究组小鼠TNF-α、IL-6、IL-22与IL-17水平降低,IL-35和可溶性Fas水平升高(P<0.05)。结肠组织病理分析结果显示,与空白组比较,模型组小鼠显示不同程度的黏膜变薄,上皮结构缺失,腺体分布无规则性,固有层充血水肿,伴大量炎症细胞浸润及毛细血管充血、淋巴管明显扩张,弥漫分布小溃疡;研究组小鼠结肠黏膜病变程度较模型组有所减轻,但部分仍可见淋巴管轻度扩张,伴少量炎症细胞浸润。菌群分析结果显示,与空白组比较,模型组和研究组小鼠肠杆菌、肠球菌、拟杆菌和梭菌的菌群值显著升高,B/E值降低(P<0.05);与模型组比较,研究组小鼠酵母菌、双歧杆菌和乳酸杆菌的菌群值明显增加,B/E值升高(P<0.05)。结论粪菌移植可改善UC小鼠结肠组织病理性损伤及小鼠肠道菌群结构,减少促炎因子的表达,促进抑炎因子的生成,调节肠道免疫平衡,促进肠黏膜修复。
Objective To analyze the therapeutic effect of fecal microbiota transplantation(FMT)on dextran sulfate sodium(DSS)induced ulcerative colitis(UC)mice and explore the relevant immunological mechanisms.Methods A total of 60 C57BL/6J mice were randomly selected,with 10 mice used for fecal collection(did not participate in the subsequent experiments),and the remaining 50 mice randomly divided into 3 groups:blank group(10 mice),which received normal diet and free access to sterile distilled water;model group and research group(20 mice each),in which ulcerative colitis(UC)mouse model was successfully established using dextran sulfate sodium(DSS).Model group and research group were allowed to freely drink a 2%dextran sulfate sodium(DSS)solution from the first day of self-grouping,to induce the formation of ulcerative colitis mouse model.After the mouse animal model was established,the model group was given saline enema,and the research group was given normal mouse fecal suspension saline mixfure enema twice,with a time interval of 3 days.On the second day after the completion of the second enema,all mice were euthanized and serum samples were collected.The expression levels of cell factors such as TNF-α,IL-6,IL-35,IL-22,IL-17 and soluble Fas in serum were determined using ELISA.The pathological morphology of the ulcerative colitis lesion in the colon tissue was observed by HE staining of the specimens,and the intestinal microbiota values and B/E values were quantitatively detected using the Japanese Oka intestinal microbiota analysis method.Results ELISA testing indicated that compared to blank group,model group and study group showed upregulation of TNF-α,IL-6,IL-17 and IL-22 levels,while IL-35 and soluble Fas levels were decreased(P<0.05).Compared to the model group,the study group showed downregulation of TNF-α,IL-6,IL-22 and IL-17 levels,with an increase in IL-35 and soluble Fas levels(P<0.05).Histopathological analysis of colon tissues showed that compared to the blank group,the model group displayed varying degrees of thinning of the mucosa,loss of epithelial structure,irregular distribution of glands,congestion and edema in the submucosal layer,along with significant infiltration of inflammatory cells,congestion of capillaries,obvious dilation of lymphatic vessels and diffuse distribution of small ulcers.The degree of colonic mucosal lesions in the study group mice was reduced compared to the model group,but partial mild dilation of lymphatic vessels and infiltration of a small amount of inflammatory cells could still be observed.Analysis of bacterial flora showed that compared to the blank group,the model group and the study group showed significantly increased levels of intestinal bacteria,enterococci,pseudomonas and clostridium,with a decreased B/E value(P<0.05).Compared to the model group,the study group showed a significant increase in the levels of yeast,bifidobacteria and lactobacilli,with an increased B/E value(P<0.05).Conclusion Fecal microbiota transplantation can improve the pathological damage of colonic tissue in UC mice and the structure of the intestinal microbiota in mice,reduce the expression of pro-inflammatory factors,promote the generation of anti-inflammatory factors,regulate intestinal immune balance and promote intestinal mucosal repair.
作者
刘扬
刘青
路明
LIU Yang;LIU Qing;LU Ming(Department of Anorectal Surgery,the First Affiliated Hospital of Xinjiang Medical University,Urumqi 830054,China)
出处
《新疆医科大学学报》
CAS
2024年第2期192-196,共5页
Journal of Xinjiang Medical University
基金
新疆维吾尔自治区自然科学基金项目(2021D01C332)。
关键词
粪菌移植
溃疡性结肠炎
免疫学
机制研究
fecal microbiota transplantation
ulcerative colitis
immunology
mechanism research
