葡聚糖硫酸钠结肠炎模型的制作方法、特点和影响因素

近几年来。尽管溃疡性结肠炎（ulcerative colitis，UC）的基础和临床研究有了较大的进展。但对进一步深入认识UC的病理生理机制和开发治疗药物．建立合适的动物模型是必不可少的。理想的UC动物模型应力求其病因、病理生理、组织形态和临床表现近似于人类UC。从20世纪60年代起。人们就开始通过应用不同动物、致炎方法和制作原理，制造出多种UC模型，但大多数模型不具备慢性复发性特点，葡聚糖硫酸钠（dextran sulfate sodium，DSS）模型则可通过改变其浓度和作用时间克服此缺陷，现国内外应用最多的是DSS诱导的UC模型。本文重点就DSSUC模型的制作方法、特点和影响因素作一综述。     

丝裂原活化蛋白激酶选择性抑制剂对葡聚糖硫酸钠结肠炎的影响

目的探讨p38丝裂原活化蛋白激酶（p38MAPK）选择性抑制剂SB203580对葡聚糖硫酸钠（DSS）诱导的小鼠结肠炎的影响。方法24只雌性BALB／C小鼠均分为三组：正常对照组、DSS结肠炎组和SB203580干预组。结肠炎组小鼠每日自由摄取5％DSS溶液．干预组小鼠在摄取5％DSS溶液72h后，每日腹腔注射SB2035801mg／kg体质量。各组小鼠每日记录疾病活动指数（DAI）评分，H-E染色观察小鼠结肠黏膜组织学改变。ELISA法测定结肠组织巾肿瘤坏死因子（TNF）-a含量。免疫组化染色观察磷酸化的活化转录因子2（P-ATF2）在结肠组织炎性细胞内的表达情况。结果正常对照组和DSS结肠炎组小鼠的DAI评分为0．224-0．09和4．81±1．08（P〈0．05），组织学评分为1．03±0．38和11．0±0．75（P〈0．05），经SB203580干预后分别为1．96±0．89和7．16±1．46，与结肠炎组小鼠比较差异有统计学意义（P00．05）。正常对照组与DSS结肠炎组小鼠的结肠匀浆TNF-a含量分别为（42．98±17．31）和（128．34±33．76）pg／ml（P〈0．05），SB203580于预后下降为（81．86±25．11）pg／ml，与结肠炎组小鼠比较差异有统计学意义（P〈0．05）。正常对照组与结肠炎组小鼠结肠组织炎性细胞P-ATF2的表达分别为（6．91±1．83）％和（81．02±12．35）％（P〈0．05），经SB203580处理后为（38．59±8．12）％，与结肠炎组小鼠比较差异有统计学意义（P00．05）。结论阻断p38MAPK信号转导通路可明显缓解DSS诱导的结肠炎。预示此信号转导通路可为溃疡性结肠炎新药研究提供有价值的靶标。     

不同浓度葡聚糖硫酸钠诱导小鼠炎症性肠病模型的实验研究

目的 探索饮用不同浓度葡聚糖硫酸钠(DSS)诱导小鼠炎症性肠病(IBD)模型构建。方法 选取SPF级雌性C57BL6小鼠40只,随机分为5组,每组8只。通过饮用不同浓度DSS,诱导IBD小鼠模型,实验组自由饮用不同浓度DSS(2.0%、2.5%、3.0%、3.5%)7 d;对照组自由饮用灭菌蒸馏水7 d。观察小鼠一般情况和结直肠病理学改变,记录结直肠长度及体质量变化,进行疾病活动指数(DAI)和肠道组织病理学评分,采用ELISA检测小鼠血清白介素-6(IL-6)水平,以评估模型诱导的效果。结果 与对照组相比,各DSS组小鼠一般情况较差。2.0%DSS组小鼠体质量下降(P <0.05),结直肠长度显著缩短,DAI评分和肠道组织病理学评分以及血清IL-6水平均显著升高(P<0.01);2.5%、3.0%、3.5%DSS组各项观察指标值差异均有统计学意义(P <0.01)。血清IL-6水平升高且与DSS浓度呈正相关。2.5%、3.0%DSS组之间小鼠体质量、结直肠长度、DAI评分、肠道组织病理学评分差异均无统计学意义(P>0.05)。与2.5%DSS组比较,3.5%D...     

葡聚糖硫酸钠致溃疡性结肠炎大鼠模型的肠道微生态

目的:采用多聚酶链反应-变性梯度凝胶电泳(polymerase chain reaction-denaturing gradient gel electrophoresis,PCR-DGGE)技术研究葡聚糖硫酸钠(dextran sulfate sodium,DSS)所致溃疡性结肠炎(ulcerative colitis,UC)模型大鼠肠道菌群的多样性和丰度,从"肠道微生态"这一全新角度为该模型提供更丰富的数据,也为深入探讨UC的发生发展提供新的实验依据.方法:26只健康♂SD大鼠随机分为空白组和UC模型组,模型组饮用4%的DSS溶液7d复制UC模型.收集两组大鼠粪便排泄物,采用PCR-DGGE法研究肠道菌群的多样性、相似性及丰度;对优势条带进行回收测序,鉴定菌种.所得结果及数据采用Quantity one、Chromas、MGAE5、SIMCA-P+及SPSS18.0等软件进行分析.结果:DSS诱导UC后7d,模型大鼠出现便血、病理形态学改变等典型的UC炎性病变特征.样本优势条带菌种鉴定显示大鼠的肠道细菌主要归属于拟杆菌门(Bacteroidetes)、厚壁菌门(Firmicutes)及变形菌门(Proteobacteria)3大类,但与空白组相比,模型组肠道菌群的丰度及多样性指数明显降低(P<0.05).菌种鉴定表明UC组乳酸杆菌(Lactobacillus sp)和毛螺科菌(Lachnospiraceae bacterium)菌种显著较少,而双酶梭菌(Clostridium bifermentans)等菌种含量明显增多(均P<0.05).     

丹参对右旋葡聚糖硫酸钠诱导小鼠结肠炎的疗效观察

目的 :评价丹参预防及治疗右旋葡聚糖硫酸钠 (DSS)结肠炎小鼠的有效性。方法 :2 0只正常小鼠随机分为两组 ,饮用 DSS7d,同时预防组用丹参 ,对照组用 0 .85 %氯化钠溶液。另 2 0只 DSS诱导的结肠炎小鼠随机分为两组 ,治疗组用丹参 ,对照组用 0 .85 %氯化钠溶液 7d。用疾病活动指数 (DAI)、组织学评分和马休斯猩红蓝(MSB)纤维素染色检测微血栓以评价疗效。结果 :丹参在预防组部分降低微血栓的形成 ,对照组 10例有 6例微血栓阳性 ,预防组 3例阳性。丹参治疗组与对照组的 DAI、直肠、横结肠组织学评分分别为 0 .4 5、0 .4 8(P>0 .0 5 ) ,1.36、1.76 (P<0 .0 5 ) ,1.35、1.6 0 (P<0 .0 5 )。结论 :丹参可能部分抑制微血栓形成和减轻 DSS结肠炎小鼠结肠炎症 ,提示丹参用于溃疡性结肠炎治疗也可能有效。     

葡聚糖硫酸钠诱导小鼠实验性结肠炎的临床和组织病理学特征研究

目的研究葡聚糖硫酸钠(DSS)诱导小鼠实验性结肠炎的临床和病理特征,以便于指导选择合适的溃疡性结肠炎(ulcerative colitis,UC)小鼠模型。方法予3%DSS溶液喂饲野生型C57BL/6成年小鼠诱导急性结肠炎模型,分别于第0天、第3天、第5天、第7天和第10天麻醉处死小鼠,取结肠观察不同时期结肠病理学特征,造模过程中连续观察并记录小鼠体质量、大便和死亡情况。结果小鼠造模第3天开始出现粪便潜血,第5天开始出现血便,第10天开始出现死亡小鼠,死亡率为30%。DSS诱导小鼠急性结肠炎模型疾病活动指数第3天后与饮用纯净水的小鼠比较明显增高(P0.05)。小鼠结肠炎造模第3天黏膜上皮细胞开始逐渐丧失,第7~10天最为严重;隐窝结构的紊乱从第3天开始发生,第7天出现固有层塌陷;炎性细胞浸润从第3天开始数量逐渐增加,第10天最为严重。DSS诱导小鼠急性结肠炎模型结肠组织病理评分第5天后与饮用纯净水的小鼠比较明显增高(P0.05)。结论 3%DSS诱导野生型C57BL/6成年小鼠急性结肠炎模型可用于UC的实验研究,第3天出现明显的炎症表现,第7天模型较理想,小鼠死亡少。     

小鼠葡聚糖硫酸钠急性溃疡性结肠炎模型的建立和评价

目的探索葡聚糖硫酸钠(dextransulfatesodium,DSS)诱导UC急性模型的浓度及时间。方法用C57BL/6小鼠自由饮用DSS溶液(3%、5%、8%)的方法建立急性UC模型,正常组饮用蒸馏水作为对照。通过疾病活动指数(DAI)和肠黏膜组织损伤指数(HI)来评价结肠的形态和组织病理学改变。结果DAI评分随着浓度(0、3%、5%和8%)的增加呈线性增加(1.50±0.53、7.71±1.38、9.14±1.03和9.71±0.61),呈浓度依赖性(组间比较P<0.05);远端结肠HI评分(0、5.93±1.65、7.00±1.07、7.75±0.46)和近端结肠HI评分(0、4.25±1.58、5.13±3.17、6.50±1.41)无浓度依赖性(仅3%组vs8%组P<0.05);死亡率随浓度的增加而升高(0、7.10%、28.60%和57.10%)。结论DSS模型的临床表现和病理表现与人类UC及其相似,是理想的UC模型。DSS模型结肠炎症程度的轻重与浓度无明显相关。采用C57BL/6小鼠饮用3%DSS(分子量为40000左右)溶液5天的方法可建立理想的UC急性模型。     

二甲肼和葡聚糖硫酸钠建立溃疡性结肠炎相关性大肠癌小鼠模型

目的建立溃疡性结肠炎相关性大肠癌的动物模型，并检测该模型诱发的不典型增生及癌变组织中β-catenin和p53的表达情况。方法单次小剂量腹腔注射二甲肼（20mg／kg）联合三循环3％葡聚糖硫酸钠自由饮用建立小鼠溃疡性结肠炎相关性大肠癌模型，HE染色观察结肠、盲肠黏膜不典型增生及癌变的发生，免疫组组织化学法观察不典型增生及癌变组织中β-catenin和p53的表达情况。结果低剂量二甲肼腹腔注射联合葡聚糖硫酸钠三个循环11只小鼠10周内共诱发4处原位癌，36处不典型增生，肿瘤主要位于远端结肠，单独二甲肼或葡聚糖硫酸钠处理无肿瘤发生。原位癌和不典型增生组织中β-catenin在细胞核，细胞浆和细胞膜中均有表达。高度不典型增生（73．3％：2＋）比低度不典型增生（30．0％：2＋）表达强。p53在原位癌和大肠典型增生组织中均无表达。结论单剂量二甲肼利葡聚糖硫酸钠合川在短期内能诱导实验小鼠结肠肿瘤发生。此模型对溃疡性结肠炎相关的人肠癌的研究可能有应用价值。     

葡聚糖硫酸钠自由饮用与定量灌胃诱导小鼠急性结肠炎模型的比较研究

背景：自由饮用葡聚糖硫酸钠（DSS）诱导的鼠类急性结肠炎模型均一性欠佳,动物死亡率较高。目的：评估2%DSS自由饮用或定量灌胃诱导的小鼠急性结肠炎模型模拟人类溃疡性结肠炎（UC）的效果和均一性。方法：予ICR小鼠2%DSS自由饮用或定量灌胃建立急性结肠炎模型,检测并比较正常对照组和两组模型小鼠的症状出现时间和频率、疾病活动指数（DAI）、DSS消耗量、死亡率、结肠长度、结肠损伤大体评分（MACD）、结肠组织病理学表现以及外周血白细胞计数和分类。结果：两组模型小鼠均出现类似人类UC的症状和组织病理学改变,结肠显著短缩,DAI、MACD以及外周血白细胞计数和中性粒细胞百分比显著高于正常对照组。与DSS自由饮用组相比,DSS定量灌胃组小鼠症状出现时间更为一致,症状出现率显著增高,动物死亡率和DSS消耗量显著减低。结论：2%DSS定量灌胃诱导的小鼠急性结肠炎模型能较稳定地模拟人类UC,均一性高,动物死亡率低。     

Attenuation of dextran sodium sulphate induced colitis in matrix metalloproteinase-9 deficient mice

INTRODUCTIONMatrix metalloproteinases(MMPs)comprise a group of zinc and calcium-dependent endopeptidases that exhibit differential proteolytic activity against extracellular matrix(ECM)proteins.Based on substrate specificity,MMPs have been classically div…     

Temporal clinical,proteomic, histological and cellular immune responses of dextran sulfate sodium-induced acute colitis

AIM To investigate the temporal clinical, proteomic, histological and cellular immune profiles of dextran sulfate sodium(DSS)-induced acute colitis.METHODS Acute colitis was induced in C57 BL/6 female mice by administration of 1%, 2% or 3% DSS in drinking water for 7 d. Animals were monitored daily for weight loss, stool consistency and blood in the stool, while spleens and colons were harvested on day 8. A time course analysis was performed in mice ingesting 3% DSS, which included colon proteomics through multiplex assay, colon histological scoring by a blinded investigator, and immune response through flow cytometry or immunohistochemistry of the spleen, mesenteric lymph node and colon.RESULTS Progressive worsening of clinical colitis was observed with increasing DSS from 1% to 3%. In mice ingesting 3% DSS, colon shortening and increase in proinflammatory factors starting at day 3 was observed, with increased spleen weights at day 6 and day 8. This coincided with cellular infiltration in the colon from day 2 to day 8, with progressive accumulation of macrophages F4/80~+, T helper CD4~+(Th), T cytotoxic CD8~+(Tcyt) and T regulatory CD25~+(Treg) cells, and progressive changes in colonic pathology including destruction of crypts, loss of goblet cells and depletion of the epithelial barrier. Starting on day 4, mesenteric lymph node and/or spleen presented with lower levels of Treg, Th and Tcyt cells, suggesting an immune cell tropism to the gut. CONCLUSION These results demonstrate that the severity of experimental colitis is dependent on DSS concentration, correlated with clinical, proteomic, histological and cellular immune response on 3% DSS.     

肝素治疗右旋葡聚糖硫酸钠诱导小鼠结肠炎的疗效观察

目的　评价肝素预防及治疗右旋葡聚糖硫酸钠 (DSS)诱导小鼠结肠炎的有效性。方法　32只小鼠中 ,16只正常小鼠分成两组 ,饮用DSS 7d的同时预防组皮下注射肝素 ,对照组皮下注射生理盐水 ;另 16只饮用DSS 7d后诱导结肠炎的小鼠分成两组 ,治疗组皮下注射肝素 ,对照组皮下注射生理盐水。用疾病活动指数、组织学评分、TNF α的表达和马休斯猩红兰 (MSB)检测微血栓评价疗效。结果　肝素在预防组降低微血栓的形成 ,对照组 8只中 4只微血栓阳性 ,预防组均阴性 (P =0 .0 38)。治疗组组织学评分、TNF α的表达明显降低 ,治疗组与对照组的直肠、横结肠组织学评分和TNF α的表达分别为 1.33和 1.85 (P <0 .0 5 ) ,0 .92和 1.6 8(P <0 .0 5 ) ,(5 .5± 3.5 ) %和 (10 .8± 4.2 ) % (P <0 .0 5 )。结论　肝素可抑制DSS诱导结肠炎小鼠血栓形成和结肠炎症 ,实验结果提示肝素用于溃疡性结肠炎治疗也可能有效     

Kefir treatment ameliorates dextran sulfate sodium-induced colitis in rats

AIM: To investigate the preventive effect of kefir on colitis induced with dextran sulfate sodium(DSS) in rats.METHODS: Twenty-four male Wistar-albino rats were randomized into four groups: normal control,kefircontrol,colitis,and kefir-colitis groups. Rats in the normal and kefir-control groups were administered tap water as drinking water for 14 d. Rats in the colitis and kefir-colitis groups were administered a 3% DSS solution as drinking water for 8-14 d to induce colitis. Rats in the kefir-control and kefir-colitis groups were administered 5 m L kefir once a day for 14 d while rats in the normal control and colitis group were administered an identical volume of the placebo(skim milk) using an orogastric feeding tube. Clinical colitis was evaluated with reference to the disease activity index(DAI),based on daily weight loss,stool consistency,and presence of bleeding in feces. Rats were sacrificed on the 15 th day,blood specimens were collected,and colon tissues were rapidly removed. Levels of myeloperoxidase(MPO),tumor necrosisfactor(TNF)-α,interleukin(IL)-10,malondialdehyde,and inducible nitric oxide synthase(i NOS) were measured in colon tissue.RESULTS: The DAI was lower in the kefir-colitis group than in the colitis group(on the 3rd and 5th days of colitis induction; P 0.01). The DAI was also significantly higher in the colitis group between days 2 and 6 of colitis induction when compared to the normal control and kefir-control groups. The DAI was statistically higher only on the 6th day in the kefircolitis group when compared to that in the normal control groups. Increased colon weight and decreased colon length were observed in colitis-induced rats. Mean colon length in the colitis group was significantly shorter than that of the kefir-control group. Kefir treatment significantly decreased histologic colitis scores(P 0.05). MPO activity in the colitis group was significantly higher than in the kefir-control group(P 0.05). Kefir treatment significantly reduced the DSS colitis-induced TNF-α increase(P 0.01). No statistically significant differences were observed among groups for IL-10 and MDA levels. Colon tissue i NOS levels in the colitis group were significantly higher than those in the control and kefir-colitis groups(P 0.05).CONCLUSION: Kefir reduces the clinical DAI and histologic colitis scores in a DSS-induced colitis model,possibly via reduction of MPO,TNF-α,and i NOS levels.     

Antibody to eosinophil cationic protein suppresses dextran sulfate sodium-induced colitis in rats

AIM: To produce an antibody against rat eosinophil cationic protein (ECP) and to examine the effects of the antibody in rats with dextran sulfate sodium (DSS)-induced colitis. METHODS: An antibody was raised against rat ECP. Rats were treated with 3% DSS in drinking water for 7 d and received the antibody or normal serum. The colons were examined histologically and correlated with clinical symptoms. Immunohistochemistry and Western blot analysis were estimated as a grade of inflammation. RESULTS: The ECP antibody stained the activated eosinophils around the injured crypts in the colonic mucosa. Antibody treatment reduced the severity of colonic ulceration and acute clinical symptoms (diarrhea and/or bloodstained stool). Body weight gain was significantly greater and the colon length was significantly longer in anti-ECP-treated rats than in normal serum-treated rats. Expression of ECP in activated eosinophils was associated with the presence of erosions and inflammation. The number of Ki-67-positive cells in the regenerated surface epithelium increased in anti-ECP-treated rats compared with normal serum-treated rats. Western blot analysis revealed reduced expression of macrophage migration inhibitory factor (MIF) in anti-ECP-treated rats. CONCLUSION: Our results indicate that treatment with ECP antibody, improved DSS-induced colitis in rats, possibly by increasing the regenerative activity of the colonic epithelium and downregulation of the immune response, and suggest that anti-ECP may promote intestinal wound healing in patients with ulcerative colitis (UC).     

Bosentan, an endothelin receptor antagonist, reduces leucocyte adhesion and inflammation in a murine model of inflammatory bowel disease

Background and aims Endothelins, a group of polyfunctional cytokines, induce the adhesion of circulating leucocytes to venous endothelium, an initial step in the pathogenesis of a cellular infiltrate in inflammatory bowel disease (IBD). The effect of bosentan, a non-selective endothelin receptor antagonist, on leucocyte adhesion and inflammation in a murine model of IBD was studied.Materials and Methods Thirty BALB/c mice were divided into three groups of 10 animals: untreated controls, chronic colitis [dextran sodium sulphate (DSS), 3% w/v for 30 days], and treatment with bosentan (30 mg/kg i.p. daily on days 26–30). On day 30, adherent and rolling leucocytes and the average rolling velocity were assessed by intravital microscopy. Clinical and histological activity of inflammation were assessed by the disease activity index and modified Dieleman score, respectively.Statistics Kruskal–Wallis test was used, followed by Dunn's method. A value of p<0.05 was considered significant.Results Compared to healthy controls, mice treated with DSS showed pronounced clinical and histological inflammation, and a higher number of rolling and adhering leucocytes in colonic submucosal venules. Therapy with bosentan significantly reduced clinical and histological inflammation. Adherent leucocyte levels were markedly lower (1.2±0.3 vs 23.7±2.8 adherent cells per 0.01 mm², p<0.05). The number of rolling leucocytes was lower but not significantly different. However, rolling velocity was significantly higher (91.5±14.0 vs 19.0±1.6 μm/s, p<0.05).Conclusions Bosentan reduces the adhesion of leucocytes in colonic submucosal venules and reduces inflammation in this mouse model of IBD. By inhibiting leucocyte adhesion, a crucial step in the recruitment of leucocytes to the inflamed tissue, bosentan is a potent therapeutic drug in this animal model. Further studies are necessary to investigate the role of bosentan as a novel drug in human IBD.C. Anthoni and R.B. Mennigen contributed equally to this work     

凝结芽孢杆菌活菌TBC169株对右旋葡聚糖硫酸钠引发大鼠溃疡性结肠炎的治疗作用

目的:研究凝结芽胞杆茵(Bacillus coagulans,BC)对右旋葡聚糖硫酸钠(DSS)引发的大鼠溃疡性结肠炎(UC)的治疗作用.方法:采用DSS引发大鼠UC,分别用10~7和10~6 CFU/mL BC、0.02g/mL柳氮磺胺吡啶(SASP)和生理盐水(NS)处理.21d后测定动物体质量、结直肠湿重、肠黏膜溃疡、糜烂点数及面积、髓过氧化物酶活性和肠道菌群培养.结果:治疗21d后,各组中大鼠的体质量增加,以BC10~7 CFU/mL增重明显(P<0.05);各治疗组中大鼠的结直肠湿重、肠溃疡糜烂点数、面积、MPO活性与模型组比较均有显著性差异(P<0.05或P<0.01或P<0.001);肠道菌群分析,造型后双歧杆菌数明显下降(P<0.05),治疗后各组的双歧杆菌数量均明显增加(P<0.01或P<0.001),以BC组和SASP组增加最明显(P<0.01或P<0.001).BC在肠道定植.结论:BC对DSS引发的大鼠UC有明显治疗作用.     

Methotrexate in ulcerative colitis: A Spanish multicentric study on clinical use and efficacy

Background

Few data are available on the efficacy of methotrexate (MTX) in ulcerative colitis (UC).

Aim

To evaluate the efficacy and safety of MTX in UC patients.

Patients and methods

UC patients who had been treated with MTX were identified from the databases of 8 Spanish IBD referral hospitals. Patients were included in the study if they received MTX for steroid dependency or steroid refractoriness. Therapeutic success was defined as the absence of UC-related symptoms, complete steroid withdrawal and no requirement of rescue therapies within the first 6 months after starting MTX.

Results

Forty patients were included, 70% treated for steroid dependency and 27% for steroid refractoriness. Thiopurines had been previously attempted in 87.5% of patients. The median dose of MTX used for induction was 25 mg (IIQ 17.5–25) weekly given parenterally in 82.5% of cases. Eighty-five percent of patients were on steroids when MTX was started. Forty-five percent of patients met criteria for therapeutic success. Initial treatment failures were mainly due to inefficacy (50%) or intolerance (36%). After a median follow-up of 28 months (IQR 22–47), 38% of patients with initial therapeutic success required new steroid courses, 22% started biological therapy, and only 1 patient required colectomy. The cumulative probability of maintaining steroid-free clinical remission was 60%, 48%, and 35% at 6, 12, and 24 months after starting MTX, respectively. Eleven patients (27.5%) experienced adverse events, leading to MTX discontinuation in only 8 of them.

Conclusions

MTX appears to be effective to maintain clinical remission in UC, at least in the short-term, with an acceptable safety profile.