这项工作是由加拿大卫生研究院（CIHR）的赠款和克隆氏病和结肠炎基金会（CCFC）加拿大支持。

1。 DSS诱导的急性结肠炎的小鼠模型<ol><li>添加葡聚糖硫酸盐钠（DSS）蒸压饮用水所需的最终浓度（1-5％）（WT /卷）（即5％的DSS解决方案，添加25毫克的DSS粉蒸压水500毫升） 。联合解决方案可以在室温下长达一周或4℃直至使用。 </li><li>在生物安全罩，倒入50毫升猎鹰管（每笼所需）股票DSS解决方案。保持股票笔芯管的解决方案，在需要的时候。 </li><li>每个鼠笼更换饮用水与DSS解决方案（50毫升猎鹰管）（持续时间将取决于对DSS的方案，用于，例如，用6-8周龄雄性C57BL / 6小鼠，我们管理5％DSS解决方案共5天）。老鼠不应该有任何其他水源的访问。对照组无DSS的蒸压饮用水。 </li><li>小鼠每天称重并记录每天消耗的直资的金额。顶部每个一瓶50毫升录制后直资水平。这是衡量每人鼠笼消耗DSS整个实验期间的近似体积。在我们的研究中，我们用5天雄性C57BL / 6小鼠5％DSS解决方案。被视为第3天早期使用这个特殊的DSS疗法显着的减肥，改变大便的一致性和粪便中血液的迹象。在DSS管理，小鼠表现出脱水和腹泻明显体重消瘦（第5天其初始体重的5-10％左右）大于20％初始体重显著的生理指标，动物或附近的端点。如果动物进入正常蓄水后5天5％DSS的，它会在7天内恢复。所有的实验应该由该机构的动物伦理委员会批准，并按照批准的动物利用协议（AUP）。 </li><li>在实验的持续时间，疾病活动指数（DAI）得分可以进行评估，评估结肠炎的临床进展。傣族是减肥的综合得分比初始体重，大便的一致性，和出血。分数的定义如下：减肥：0（无损失），1（1-5％），2（5-10％），3（10-20％）和4（&gt; 20％），大便的一致性： 0（正常），2（便溏），4（腹泻）;出血：0（无血缘），1（Hemoccult阳性），2（Hemoccult积极和视觉的颗粒出血）和4（严重出血，血液周围肛门）。戴可取得每天在DSS的治疗时间。 </li><li>在时间点的选择，权衡和牺牲老鼠。吸入异氟醚或另经该机构的动物设施批准方法，老鼠可以安乐死颈椎脱位。 </li></ol> 2。收集结肠组织样品<ol><li>揭露动物的腹侧面和腹部面积70％的乙醇溶液与湿。在这一点上，请参考表1和一个音符NY直肠出血（血在肛门口目前），或在每个动物的直肠脱垂的迹象。 </li><li>使用标准的解剖剪刀切开腹中线切口腹部。 </li><li>找到结肠癌和断面接近大肠癌保证金尽可能免费远端结肠的结肠。 </li><li>小心缓慢地拉出整个结肠，分离它从周围的肠系膜。 </li><li>在colonocecal距断面免费的结肠近端结肠。可以去除粪便通过仔细挤压它使用一对弯曲的镊子/钳连接到3或5毫升的注射器或使用灌胃针，用无菌PBS冲洗结肠。使用全结肠，评估损坏（见3.1节） </li><li>可以通过组织作病理分析及其他分析取样注意哪些领域的样本是从削减0.5厘米，1.0厘米长的结肠片段（即近端，中间，或远端）。 </li><li>组织样本要使用用于检测可单独放置于1.5 mL Eppendorf管和液氮冷冻和储存在-70 ° C，直到使用</li></ol> 3。结肠炎严重程度的评估<ol><li>宏观或疾病的严重程度评分是不偏不倚的观察员使用一个先前公布的计分制（见表1）末期评估。9大便的一致性，可以通过使用一对镊子和紧迫的粪便，以确定一致性评估。要确定为血液在粪便中的得分，注意粪便的颜色（即黑便与浅棕色大便），并进一步验证使用Hemoccult的测试。使用计分制度，确定每个条件的得分。每个动物的宏观的最终得分是每一个人的得分总和。 </li><li>为了评估结肠炎严重程度的病理损害，降低结肠的小片段（0.5厘米），纸巾盒的地方，淹没在缓冲10％福尔马林溶液。准备5μm的石蜡包埋的截面与苏木/ eoxin（H＆E）和污渍的部分，使用适当的程序。科隆片段，可以采取从近，中期结肠癌，或结肠的远端部分。 </li><li> H＆E染色结肠组织部分都取得了一个盲以下措施以前出版系统观察员：地穴架构（正常，0 - 严重损失整个隐窝，3地穴失真），程度的炎性细胞浸润（正常，0 - 密集的炎性浸润，3），肌增厚（隐窝基地位于粘膜肌层，0 - 明显的肌增厚目前，3），杯状细胞耗竭（缺席，0 - 目前，1）和隐窝脓肿（缺席，0 - 至今1）7，组织学损伤评分是每一个人得分的总和。应该指出，不像人类UC，隐窝脓肿这种模式的特点，都是世所罕见;微观溃疡也属罕见。如果有多个结肠节染色组织学评分，类似部分之间应使用，以确定最后的比分为每个区域（​​即近端结肠和远端结肠组织学评分的组织学评分）。 </li></ol> 4。准备检测试剂的库存解决方案<ol><li>准备一个50毫米的磷酸钾缓冲溶液加入溶液B（K 2 HPO 4，二元磷酸钾8.7克，1升的dh 2）解决方案（KH 2 PO 4，二氢磷酸钾6.8克1L 生署2 O），直到pH值6.0的实现。剩余的解决方案，可存放在冰箱（2-8℃），直到将来使用。 </li><li>准备加入到磷酸钾缓冲液1升（50毫米，pH值= 6.0）5克HTAB十六烷基三甲基溴化铵（HTAB）缓冲区。轻轻地热溶解并储存在2-8℃直至使用。必要时，热重新溶解。 </li><li>准备组织同质化蛋白质分析裂解液加入10毫升的1M的Tris -盐酸（pH = 8.0的），5M氯化钠6毫升和2毫升TRITON X - 100至182毫升无菌蒸馏水。 TRITON X - 100是非常粘稠，在室温下，因此，应轻轻回暖之前使用。准备裂解液可存放在-20 ° C，直到使用。 </li></ol> 5。用于检测的样品制备<ol><li> MPO的分析样品制备。 <ol><li>取出样品从-70℃，置于冰上。记录取出后，使用到一个2ML的Eppendorf安全锁的离心管（或任何可以用于匀浆管）弯曲的forcep /镊子和地方的任何可见的粪便或脂肪每个样品的重量。样品应保留在任何时候都冰。重要的是要注意类似的结肠片段，应该从每个生物复制（即远端部分的唯一或近端部分）使用。 </li><li>珠匀浆每个样品管。 </li><li>添加适量HTAB BUFFER根据组织重量。如果组织重量小于25毫克，添加比例的12.5mg/mL缓冲区，如果组织的重量是25-50之间，在25mg/mL的比例添加。如果组织重量大于50，在50mg/mL的比例添加缓冲区。 </li><li>均质同为4分30赫兹的组织匀浆。如果组织不充分匀浆重复。 </li><li>卸下珠匀浆和离心6分钟（13400 XG，4 ° C）的解决方案。 </li><li>收集上清并丢弃所产生的颗粒。上清可存放于-70℃直至使用。 </li></ol></li><li>因子分析的样品制备。 <ol><li>重复步骤5.1.1。 5.1.2。 </li><li>准备裂解液10毫升加入50μL蛋白酶抑制剂的鸡尾酒（PIC），。 </li><li>添加1毫升PIC和裂解缓冲液，以每个样品的重量，不论。 </li><li>均质5分钟在30 Hz。如果组织不充分匀浆重复。 </li><li>删除匀浆珠和离心机soluti3300小工5分钟</li><li>收集上清并丢弃所产生的颗粒。上清可存放于-70℃直至使用。 </li></ol></li></ol> 6。炎症标志物的定量<ol><li> MPO活性测定<ol><li> O - dianisidine盐酸盐（O - dianisidine）解决方案相结合，16.7毫克dianisidine O -盐酸盐，DH 2 O 90毫升，10毫升磷酸钾缓冲准备。该解决方案应准备新鲜每检测。 </li><li>添加到一个96孔板7μL组织匀浆一式三份（在第5.1节准备）。 </li><li>添加50μL稀释的H 2 O 2（4μL30％H 2 O 2稀释于96μL生署2 O）O - dianisidine混合物。 </li><li>使用多道移液器添加200μL的O - dianisidine每个井含H 2 O 2的混合物。 </li><li>在一个分光光度计使用的450纳米测量吸光度tometer。三读以30秒的时间间隔。 </li><li>计算MPO活性。 MPO活性测定的MPO / mg组织，一个单位MPO的定义是需要降解的H 2 O在室温下每分钟2 1μmoL金额单位（U）。考虑，MPO的一个单位（U）= 1μmoL的H 2 O 2分裂和H 2 O 2，1μmoL给出了一个1.13 × 10 -2纳米/分钟的吸光度变化，确定MPO的单位，在每个样品作为光吸收变化[ΔA（T 2 - T 1）] /Δmin×（1.13 × 10 -2）。为了得到每毫克的组织单位，组织：缓冲区的比例。例如，如果一个组织：缓冲比率为50毫克/毫升，在7μL匀浆，有0.35毫克组织。因此，每毫克组织单位，除以0.35 MPO的单位。下面包含一个样本计算吸光度值（NM）（一式三份，样品已添加）： </li></ol></li></ol><table border="1"><tbody><tr><td> 样品 </td><td colspan="3"> 时间0秒 </td><td colspan="3"> 时间30秒 </td><td colspan="3"> 时间为60秒 </td></tr><tr><td>一</td><td> 1 </td><td> 2 </td><td> 3 </td><td> 1&#39; </td><td> 2&#39; </td><td> 3&#39; </td><td> 1“ </td><td> 2 &#39;&#39; </td><td> 3“ </td></tr><tr><td></td><td> 0.048 </td><td> 0.048 </td><td> 0.051 </td><td> 0.061 </td><td> 0.061 </td><td> 0.065 </td><td> 0.074 </td><td> 0.073 </td><td> 0.078 </td></tr></tbody></table><ol class="lroman"><li>平均在时间0秒=（0.048 + 0.048 + 0.051）/ 3 = 0.049纳米</li><li>平均时间30秒= 0.0623 nm的</li><li>平均时间为60秒= 0.075纳米</li><li>吸光度（ΔA）的变化，从0到30秒/毫克组织（假设50毫克/毫升组织：缓冲区的比例）= [（0.0623-0.049）/（1.13 × 10 -2）] / 0.35 = 3.363 </li><li>在吸收变化ANCE（ΔA）从30到60秒/ mg组织= 3.211 </li><li> * MPO活性（U / mg组织）=平均ΔA（0-30）和ΔA（30-60）= 3.287 </li></ol><ol start="2"><li>促炎性细胞因子的定量用ELISA <ol><li>细胞因子（IL -1β，IL - 6和TNF -α）水平确定使用市售酶联免疫吸附试验（ELISA）试剂盒（Quantikine小鼠; R＆D系统）。 </li><li>从每个ELISA的吸光度值是归使用布拉德福德蛋白检测每个样品分别表示单位PG /毫克蛋白质。 </li></ol></li></ol> 7。代表性的成果当局会适当的DSS方案诱导小鼠急性结肠炎。在DSS的治疗期间，戴可用于评估和评价疾病的临床进展。与DSS处理的动物将呈现显著相比，其初始权重的重量损失，稀便和粪便出血（ 图1）。牺牲和结肠癌检查后，结肠炎的严重程度是宏观的基础上缩短结肠长度，结肠出血，大便出血，大便的一致性松动，取得，与直肠出血的迹象相比，只用清水处理的控制 （图2及表1）。 H＆E染色结肠组织样品的横截面将DSS的治疗与处理过的水控制冒号（ 图3）有较高的组织学评分。为了进一步刻画DSS治疗小鼠的炎症程度，MPO活性可以从同质化的结肠组织样品进行评估。 DSS的治疗冒号将高于MPO活性控制（ 图4）。此外，这是与水平的提高，促炎性细胞因子（IL -1β，IL - 6，TNF -α）（图5）。 <img alt="图1。" src="/files/ftp_upload/3678/3678fig1.jpg" /><br/&gt; 图1男，C57BL / 6小鼠分别给予5％，在5天饮用水直资。每个动物每天戴分数评估，平均每天为每个组（平均± SEM，N = 4只/组）。 <img alt="图2。" src="/files/ftp_upload/3678/3678fig2.jpg" /> 图2。C57BL / 6小鼠分别给予5天的饮用水中的5％直资。对照组接受无DSS的水。宏观损伤评分/疾病的严重程度评分盲目评估后5天DSS诱导的结肠炎。接受直接资助计划从小鼠中分离的冒号，有较高的宏观损伤分数（直肠出血，直肠脱垂，腹泻，结肠出血），表明疾病的严重程度更大（平均± SEM，N = 4只/组）。 <img src="/files/ftp_upload/3678/3678fig3.jpg" alt="图3" /> 图3。C57BL / 6小鼠分别给予5％的饮用水DSS解决方案，以诱发结肠炎。对照组小鼠<html>没有直接资助计划收到的水。 （一）组织学评分盲目取得使用H＆E染色结肠组织在第5天，直资后管理收集的部分。 （二）DSS处理的样品展示（三）控制（平均± SEM，N = 4只/组）相比，更多的组织学损伤（细胞浸润，杯状细胞消耗更多，更大的失真/损坏隐窝架构）。 （二），（三），星号（*）表示杯状细胞耗竭和歪曲隐窝建筑面积;数字符号（＃）表示细胞浸润。 <img alt="图4。" src="/files/ftp_upload/3678/3678fig4.jpg" /> 图4。小鼠牺牲5天DSS和结肠组织样品后管理，收集评估MPO活性。 DSS诱导的结肠炎的严重程度与MPO活性高的水平相比，对照组（平均± SEM，N = 4只/组）。 _upload/3678/3678fig5.jpg“/&gt; 亲炎症细胞因子，如IL -1β，IL - 6，TNF -α水平的提高（平均± SEM，N = 4的小鼠也与图5中除了MPO水平较高，DSS诱导的结肠炎严重程度。 /组）。 <table border="1"><tbody><tr><td> 分数 </td><td> 直肠出血 </td><td> 直肠脱垂 </td><td> 大便性状 </td><td> 血 </td></tr><tr><td> 0 </td><td>没有</td><td>没有</td><td>正常</td><td>正常</td></tr><tr><td> 1 </td><td>红</td><td>脱垂的迹象</td><td>软</td><td>红</td></tr><tr><td> 2 </td><td>暗红色</td><td>清除脱垂</td><td>非常柔软</td><td>暗红色</td></tr><tr><td> 3 </td><td>毛出血</td><td>广泛脱垂</td><td>腹泻</td><td>黑色</td></tr></tbody></t能&gt; 宏观/疾病的严重程度分数表1。

<ol>
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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+vagus+nerve:+a+tonic+inhibitory+influence+associated+with+inflammatory+bowel+disease+in+a+murine+model.">The vagus nerve: a tonic inhibitory influence associated with inflammatory bowel disease in a murine model.</a> Gastroenterology. 131, 1122-1130 (2006).</li><li>Okayasu, I., Hatakeyama, S., Yamada, M., Ohkusa, T., Inagaki, Y., Nakaya, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+method+in+the+induction+of+reliable+experimental+acute+and+chronic+ulcerative+colitis+in+mice.">A novel method in the induction of reliable experimental acute and chronic ulcerative colitis in mice.</a> Gastroenterology. 98, 694-702 (1990).</li><li>Solomon, L., Mansor, S., Mallon, P., Donnelly, E., Hoper, M., Loughrey, M., Kirk, S., Gardiner, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dextran+sulphate+sodium+(DSS)+model+of+colitis:+an+overview.">The dextran sulphate sodium (DSS) model of colitis: an overview.</a> Comparative Clinical Pathology. 19, 235-239 (2010).</li><li>Mähler, M., Bristol, I. J., Leiter, E. H., Workman, A. E., Birkenmeier, E. H., Elson, C. O., Sundberg, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+susceptibility+of+inbred+mouse+strains+to+dextran+sulfate+sodium-induced+colitis.">Differential susceptibility of inbred mouse strains to dextran sulfate sodium-induced colitis.</a> Am. J. Physiol. 274, G544-G551 (1998).</li><li>Hans, W., Scholmerich, J., Gross, V., Falk, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+the+resident+intestinal+flora+in+acute+and+chronic+dextran+sulfate+sodium-induced+colitis+in+mice.">The role of the resident intestinal flora in acute and chronic dextran sulfate sodium-induced colitis in mice.</a> Eur. J. Gastroenterol. Hepatol. 12, 267-273 (2000).</li><li>Rath, H. C., Schultz, M., Freitag, R., Dieleman, L. A., Li, F., Linde, H., Schölmerich, J., Sartor, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+subsets+of+enteric+bacteria+induce+and+perpetuate+experimental+colitis+in+rats+and+mice.">Different subsets of enteric bacteria induce and perpetuate experimental colitis in rats and mice.</a> Infect. Immun. 69, 2277-2285 (2001).</li><li>Ghia, J. E., Li, N., Wang, H., Collins, M., Deng, Y., El-Sharkawy, R. T., Côté, F., Mallet, J., Khan, W. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serotonin+has+a+key+role+in+pathogenesis+of+experimental+colitis.">Serotonin has a key role in pathogenesis of experimental colitis.</a> Gastroenterology. 137, 1649-1660 (2009).</li><li>Elson, C. O., Beagley, K. W., Sharmanov, A. T., Fujihashi, K., Kiyono, H., Tennyson, G. S., Cong, Y., Black, C. A., Ridwan, B. W., McGhee, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hapten-induced+model+of+murine+inflammatory+bowel+disease:+mucosa+immune+responses+and+protection+by+tolerance.">Hapten-induced model of murine inflammatory bowel disease: mucosa immune responses and protection by tolerance.</a> J. Immunol. 157, 2174-2185 (1996).</li><li>Dieleman, L. A., Ridwan, B. U., Tennyson, G. S., Beagley, K. W., Bucy, R. P., Elson, C. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dextran+sulfate+sodium-induced+colitis+occurs+in+severe+combined+immunodeficient+mice.">Dextran sulfate sodium-induced colitis occurs in severe combined immunodeficient mice.</a> Gastroenterology. 107, 1643-1652 (1994).</li></ol>

没有利益冲突的声明。

DSS结肠炎是广泛使用的一种化学诱导肠道炎症模型。在这个模型中，小鼠的饮用水辅以决策支持系统，这被认为是有毒的肠上皮细胞，破坏粘膜屏障的完整性。10 DSS的管理引起的一种急性结肠炎，大便溏，大便出血，与粒细胞浸润。10在直资管理，结肠炎通常伴有明显消瘦和粪便中存在的血液，它可以由粪便隐血试验评估。5.10,11收集结肠组织样品后，结肠炎的严重程度可...

<table border="1"><tbody><tr><td> 试剂/设备名称： </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> Eppendorf公司安全锁的离心管（2ML） </td><td> Eppendorf公司</td><td> 0030 120.094 </td><td></td></tr><tr><td>百特EL808吸收酶标仪</td><td>百特</td><td> EL808 </td><td></td></tr><tr><td>葡聚糖硫酸盐钠盐试剂级（分子量36,000-50,000大） </td><td> MP生物医学</td><td> 160110 </td><td></td></tr><tr><td> GEN5（软件） </td><td>百特</td><td>版本1.10.8 </td><td></td></tr><tr><td>十六烷基溴化（HTAB） </td><td> Sigma - Aldrich公司</td><td> H5882 - 100G </td><td></td></tr><tr><td>过氧化氢，30％（重量）在水溶液中</td><td> Sigma - Aldrich公司</td><td> 216763 </td><td>保存在2-8 ° C </td></tr><tr><td>微孔PL吃了96井平底</td><td> Sarstedt </td><td> 82.1581 </td><td>一次性使用</td></tr><tr><td> O - Dianisidine </td><td> Sigma - Aldrich公司</td><td> D - 3252 </td><td>光敏感。保存在2-8 ° C </td></tr><tr><td>磷酸钾，二元</td><td>卡利登</td><td> 6620-1 </td><td></td></tr><tr><td>磷酸钾，磷酸二氢</td><td> EMD化学品</td><td> PX1565 - 1 </td><td></td></tr><tr><td>蛋白酶抑制剂的鸡尾酒</td><td> Sigma - Aldrich公司</td><td> P8340 </td><td>贮存于-20 ° C </td></tr><tr><td> TRITON X - 100 </td><td> Sigma - Aldrich公司</td><td> T8787 </td><td></td></tr><tr><td>碳化钨珠组织Lyser II </td><td> QUIAGEN </td><td> 69997 </td><td></td></tr></tbody></table>

investigating intestinal inflammation in dss-induced model of ibd

炎症性肠病（IBD）的涵盖了肠道病症的范围，其中最常见的是溃疡性结肠炎（UC）和克罗恩病（CD）。 UC和CD，在结肠，产生类似的症状轮廓包括腹泻，直肠出血，腹痛，体重减轻（1）尽管鸡传染性法氏囊病的发病机制尚不清楚，它被描述为一种多因素疾病，同时涉及遗传和环境的组成部分。 有纷繁多变的结肠炎动物模型类似于鸡传染性法氏囊病的几个特点。结肠炎范围内的动物模型，从自发产生的，在某些物种敏感菌那些需要特定的浓度结肠炎诱导的化学品，如葡聚糖硫酸盐钠（DSS），管理。化学诱导的肠道炎症模型鸡传染性法氏囊病中最常用的最好的描述的模型。 ADMINI饮用水中的决策支持系统的习作产生急性或慢性结肠炎，根据管理协议。DSS展品重量损失，便溏或腹泻的迹象的动物， 有时与直肠出血的证据。4,5在这里，我们描述的方法结肠炎的发展和由此产生的炎症反应的特点可以下列管理决策支持系统的。这些方法包括苏木/ HE染色结肠部分的组织学分析，促炎性细胞因子的测量，测定髓过氧化物酶（MPO）活性，可作为替代标记炎症6。 在疾病状态下的炎症反应的程度进行评估临床症状，或由粘膜组织的组织学改变。结肠组织学损害是通过使用计分制度，认为地穴建筑，炎性细胞浸润，肌肉吨损失评估hickening，杯状细胞消耗和隐窝脓肿7定量，促炎性细胞因子与急性发炎的特性，如白细胞介素（IL）-1β，IL - 6和肿瘤坏死因子（TNF）-α，水平可确定使用传统的ELISA方法。此外，MPO活性，可使用比色法测量和炎症指数。8 在实验性结肠炎，疾病的严重程度往往与MPO活性和促炎性细胞因子的水平较高的增长。结肠炎的严重程度和炎症相关的损害，可以通过检查大便的一致性和出血，除了评估使用肠道苏木/ HE染色结肠组织切片的病理组织学状态评估。可用于结肠组织碎片，以确定MPO活性及细胞因子的产生。两者合计，这些措施可以用来评估肠道炎症反应实验性结肠炎动物模型。

Watch this Scientific Journal Video about Investigating Intestinal Inflammation in DSS-induced Model of IBD at JoVE.com

Investigating Intestinal Inflammation in DSS-induced Model of IBD

发炎性肠道疾病的实验模型使我们能够研究与发病机制相关的复杂的先天免疫和适应性免疫反应。使用组织学评分，量化促炎性细胞因子和髓过氧化物酶活性，就可以开始评估在炎症性肠病看到这些答复。

调查在DSS诱导的鸡传染性法氏囊病模型的肠道炎症

Inflammatory bowel disease (IBD) encompasses a range of intestinal pathologies, the most common of which are ulcerative colitis (UC) and Crohn's Disease ...

investigating-intestinal-inflammation-in-dss-induced-model-of-ibd

Research

JoVE Journal

Biology

66.5K 次观看.  McMaster University. 发炎性肠道疾病的实验模型使我们能够研究与发病机制相关的复杂的先天免疫和适应性免疫反应。使用组织学评分，量化促炎性细胞因子和髓过氧化物酶活性，就可以开始评估在炎症性肠病看到这些答复。

视频: Investigating Intestinal Inflammation in DSS-induced Model of IBD

Catheterization of Intestinal Loops in Ruminants

The intestine is a complex structure that is involved not only in absorption of nutrients, but also acts as a barrier between the individual and the outside world. As such, the intestine plays a pivotal role in immunosurveillance and protection from enteric pathogens. Investigating intestinal physiology and immunology commonly employs 'intestinal loops' as an experimental model. The majority of these loop models are non-recovery surgical procedures that study short-term (&lt;24 hr) changes in the intestine (1-3). We previously created a recovery intestinal loop model to specifically measure long-term (&lt;6 mo) immunological changes in the intestine of sheep following exposure to vaccines, adjuvants, and viruses (4). This procedure localized treatments to a specific 'loop', allowing us to sample this area of the intestine. A significant drawback of this method is the single window of opportunity to administer treatments (i.e. at the time of surgery). Furthermore, samples of both the intestinal mucosa and luminal contents can only be taken at the termination of the project. Other salient limitations of the above model are that the surgical manipulation and requisite post-operative measures (e.g. administration of antibiotics and analgesics) can directly affect the treatment itself and/or alter immune function, thereby confounding results. Therefore, we modified our intestinal loop model by inserting long-term catheters into the loops. Sheep recover fully from the procedure, and are unaffected by the exteriorized catheters. Notably, the establishment of catheters in loops allows us to introduce multiple treatments over an extended interval, following recovery from surgery and clearance of drugs administered during surgery and the post-operative period.

We describe a novel surgical method for catheterizing 'intestinal loops' within the ileum of sheep. Once animals have recovered from surgery and have cleared antibiotics and analgesics, multiple treatments can be deposited directly in loops via the catheters.

导尿在反刍动物的肠袢

The intestine is a complex structure that is involved not only in absorption of nutrients, but also acts as a barrier between the individual and the ...

科研

生物学

Rat Mesentery Exteriorization: A Model for Investigating the Cellular Dynamics Involved in Angiogenesis

Microvacular network growth and remodeling are critical aspects of wound healing, inflammation, diabetic retinopathy, tumor growth and other disease conditions1, 2. Network growth is commonly attributed to angiogenesis, defined as the growth of new vessels from pre-existing vessels. The angiogenic process is also directly linked to arteriogenesis, defined as the capillary acquisition of a perivascular cell coating and vessel enlargement. Needless to say, angiogenesis is complex and involves multiple players at the cellular and molecular level3. Understanding how a microvascular network grows requires identifying the spatial and temporal dynamics along the hierarchy of a network over the time course of angiogenesis. This information is critical for the development of therapies aimed at manipulating vessel growth.
The exteriorization model described in this article represents a simple, reproducible model for stimulating angiogenesis in the rat mesentery. It was adapted from wound-healing models in the rat mesentery4-7, and is an alternative to stimulate angiogenesis in the mesentery via i.p. injections of pro-angiogenic agents8, 9. The exteriorization model is attractive because it requires minimal surgical intervention and produces dramatic, reproducible increases in capillary sprouts, vascular area and vascular density over a relatively short time course in a tissue that allows for the two-dimensional visualization of entire microvascular networks down to single cell level. The stimulated growth reflects natural angiogenic responses in a physiological environment without interference of foreign angiogenic molecules. Using immunohistochemical labeling methods, this model has been proven extremely useful in identifying novel cellular events involved in angiogenesis. Investigators can readily correlate the angiogenic metrics during the time course of remodeling with time specific dynamics, such as cellular phenotypic changes or cellular interactions4, 5, 7, 10, 11.

This article describes a simple model for stimulating angiogenesis in the rat mesentery. The model produces dramatic increases in capillary sprouting, vascular area and vascular density over a relatively short time course in a tissue that allows en face visualization of entire microvascular networks down to the single cell level.

大鼠肠系膜外化：调查涉及在血管生成细胞动力学模型

Microvacular network growth and remodeling are critical aspects of wound healing, inflammation, diabetic retinopathy, tumor growth and other disease ...

A Protocol for Lentiviral Transduction and Downstream Analysis of Intestinal Organoids

Intestinal crypt-villus structures termed organoids, can be kept in sustained culture three dimensionally when supplemented with the appropriate growth factors. Since organoids are highly similar to the original tissue in terms of homeostatic stem cell differentiation, cell polarity and presence of all terminally differentiated cell types known to the adult intestinal epithelium, they serve as an essential resource in experimental research on the epithelium. The possibility to express transgenes or interfering RNA using lentiviral or retroviral vectors in organoids has increased opportunities for functional analysis of the intestinal epithelium and intestinal stem cells, surpassing traditional mouse transgenics in speed and cost. In the current video protocol we show how to utilize transduction of small intestinal organoids with lentiviral vectors illustrated by use of doxycylin inducible transgenes, or IPTG inducible short hairpin RNA for overexpression or gene knockdown. Furthermore, considering organoid culture yields minute cell counts that may even be reduced by experimental treatment, we explain how to process organoids for downstream analysis aimed at quantitative RT-PCR, RNA-microarray and immunohistochemistry. Techniques that enable transgene expression and gene knock down in intestinal organoids contribute to the research potential that these intestinal epithelial structures hold, establishing organoid culture as a new standard in cell culture.

In this video protocol we give a step by step explanation of lentiviral transduction in organoids of primary intestinal epithelium and of processing and downstream analysis of these cultures by quantitative RT-PCR, RNA-microarray and immunohistochemistry.

协议的慢病毒转导与肠组织体下游分析

Intestinal crypt-villus structures termed organoids, can be kept in sustained culture three dimensionally when supplemented with the appropriate growth ...

A Mouse Model of Intestinal Partial Obstruction

Intestinal obstructions, that impede or block peristaltic movement, can be caused by abdominal adhesions and most gastrointestinal (GI) diseases including tumorous growths. However, the cellular remodeling mechanisms involved in, and caused by, intestinal obstructions are poorly understood. Several animal models of intestinal obstructions have been developed, but the mouse model is the most cost/time effective. The mouse model uses the surgical implantation of an intestinal partial obstruction (PO) that has a high mortality rate if it is not performed correctly. In addition, mice receiving PO surgery fail to develop hypertrophy if an appropriate blockade is not used or not properly placed. Here, we describe a detailed protocol for PO surgery which produces reliable and reproducible intestinal obstructions with a very low mortality rate. This protocol utilizes a surgically placed silicone ring that surrounds the ileum which partially blocks digestive movement in the small intestine. The partial blockage makes the intestine become dilated due to the halt of digestive movement. The dilation of the intestine induces smooth muscle hypertrophy on the oral side of the ring that progressively develops over 2 weeks until it causes death. The surgical PO mouse model offers an in vivo model of hypertrophic intestinal tissue useful for studying pathological changes of intestinal cells including smooth muscle cells (SMC), interstitial cells of Cajal (ICC), PDGFR&#945;+, and neuronal cells during the development of intestinal obstruction.

Intestinal obstructions are a partial or complete blockage of the intestine that can cause severe abdominal pain, nausea, vomiting, and preventing the passage of stool. This procedure for creating intestinal partial obsructions in mice is reliable in studying the mechanisms underlying pathological cell growth and death in the intestine.

小肠部分梗阻的小鼠模型

Intestinal obstructions, that impede or block peristaltic movement, can be caused by abdominal adhesions and most gastrointestinal (GI) diseases including ...

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A)

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer progression. How the chromosomal location and function of a centromere (i.e., centromere identity) are determined and participate in accurate chromosome segregation is a fundamental question. CENP-A is proposed to be the non-DNA indicator (epigenetic mark) of centromere identity, and CENP-A ubiquitylation is required for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability.
Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-CENP-A K124R mutant suggesting that ubiquitylation at a different lysine is induced because of the EYFP tagging in the CENP-A K124R mutant protein. Lysine 306 (K306) ubiquitylation in EYFP-CENP-A K124R was successfully identified, which corresponds to lysine 56 (K56) in CENP-A through mass spectrometry analysis. A caveat is discussed in the use of GFP/EYFP or the tagging of high molecular weight protein as a tool to analyze the function of a protein. Current technical limit is also discussed for the detection of ubiquitylated bands, identification of site-specific ubiquitylation(s), and visualization of ubiquitylation in living cells or a specific single cell during the whole cell cycle.
The method of mass spectrometry analysis presented here can be applied to human CENP-A protein with different tags and other centromere-kinetochore proteins. These combinatory methods consisting of several assays/analyses could be recommended for researchers who are interested in identifying functional roles of ubiquitylation.

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A EYFP-CENP-A

CENP-A ubiquitylation is an important requirement for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability. Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A) protein.

质谱分析，用于识别 EYFP 标记 CENP-A （EYFP-CENP-A） 的泛化

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer ...

Organoid-Derived Epithelial Monolayer: A Clinically Relevant In Vitro Model for Intestinal Barrier Function

In the past, intestinal epithelial model systems were limited to transformed cell lines and primary tissue. These model systems have inherent limitations as the former do not faithfully represent original tissue physiology, and the availability of the latter is limited. Hence, their application hampers fundamental and drug development research. Adult stem-cell-based organoids (henceforth referred to as organoids) are miniatures of normal or diseased epithelial tissue from which they are derived. They can be established very efficiently from different gastrointestinal (GI) tract regions, have long-term expandability, and simulate tissue- and patient-specific responses to treatments in vitro. Here, the establishment of intestinal organoid-derived epithelial monolayers has been demonstrated along with methods to measure epithelial barrier integrity, permeability and transport,&#160;antimicrobial protein secretion, as well as histology. Moreover, intestinal organoid-derived monolayers can be enriched with proliferating stem and transit-amplifying cells as well as with key differentiated epithelial cells. Therefore, they represent a model system that can be tailored to study the effects of compounds on target cells and their mode of action. Although organoid cultures are technically more demanding than cell lines, once established, they can reduce failures in the later stages of drug development as they truly represent in vivo epithelium complexity and interpatient heterogeneity.

Here, we describe the preparation of human organoid-derived intestinal epithelial monolayers for studying intestinal barrier function, permeability, and transport. As organoids represent original epithelial tissue response to external stimuli, these models combine the advantages of expandability of cell lines and the relevance and complexity of primary tissue.

类器官来源的上皮单层：临床相关的肠道屏障功能体外模型

In the past, intestinal epithelial model systems were limited to transformed cell lines and primary tissue. These model systems have inherent limitations ...

Intestinal Epithelial Regeneration in Response to Ionizing Irradiation

The intestinal epithelium consists of a single layer of cells yet contains multiple types of terminally differentiated cells, which are generated by the active proliferation of intestinal stem cells located at the bottom of intestinal crypts. However, during events of acute intestinal injury, these active intestinal stem cells undergo cell death. Gamma irradiation is a widely used colorectal cancer treatment, which, while therapeutically efficacious, has the side effect of depleting the active stem cell pool. Indeed, patients frequently experience gastrointestinal radiation syndrome while undergoing radiotherapy, in part due to active stem cell depletion. The loss of active intestinal stem cells in intestinal crypts activates a pool of typically quiescent reserve intestinal stem cells and induces dedifferentiation of secretory and enterocyte precursor cells. If not for these cells, the intestinal epithelium would lack the ability to recover from radiotherapy and other such major tissue insults. New advances in lineage-tracing technologies allow tracking of the activation, differentiation, and migration of cells during regeneration and have been successfully employed for studying this in the gut. This study aims to depict a method for the analysis of cells within the mouse intestinal epithelium following radiation injury.

The gastrointestinal tract is one of the most sensitive organs to injury upon radiotherapeutic cancer treatments. It is simultaneously an organ system with one of the highest regenerative capacities following such insults. The presented protocol describes an efficient method to study the regenerative capacity of the intestinal epithelium.

响应电离照射的肠上皮再生

The intestinal epithelium consists of a single layer of cells yet contains multiple types of terminally differentiated cells, which are generated by the ...

肠道炎症对已建立的小鼠结肠样的上皮影响

Studying the Epithelial Effects of Intestinal Inflammation In Vitro on Established Murine Colonoids

The intestinal epithelium plays an essential role in human health, providing a barrier between the host and the external environment. This highly dynamic cell layer provides the first line of defense between microbial and immune populations and helps to modulate the intestinal immune response. Disruption of the epithelial barrier is a hallmark of inflammatory bowel disease (IBD) and is of interest for therapeutic targeting. The 3-dimensional colonoid culture system is an extremely useful in vitro model for studying intestinal stem cell dynamics and epithelial cell physiology in IBD pathogenesis. Ideally, establishing colonoids from the inflamed epithelial tissue of animals would be most beneficial in assessing the genetic and molecular influences on disease. However, we have shown that in vivo epithelial changes are not necessarily retained in colonoids established from mice with acute inflammation. To address this limitation, we have developed a protocol to treat colonoids with a cocktail of inflammatory mediators that are typically elevated during IBD. While this system can be applied ubiquitously to various culture conditions, this protocol emphasizes treatment on both differentiated colonoids and 2-dimensional monolayers derived from established colonoids. In a traditional culture setting, colonoids are enriched with intestinal stem cells, providing an ideal environment to study the stem cell niche. However, this system does not allow for an analysis of the features of intestinal physiology, such as barrier function. Further, traditional colonoids do not offer the opportunity to study the cellular response of terminally differentiated epithelial cells to proinflammatory stimuli. The methods presented here provide an alternative experimental framework to address these limitations. The 2-dimensional monolayer culture system also offers an opportunity for therapeutic drug screening ex vivo. This polarized layer of cells can be treated with inflammatory mediators on the basal side of the cell and concomitantly with putative therapeutics apically to determine their utility in IBD treatment.

作者聚焦：研究体外肠道炎症对已建立的小鼠结肠样蛋白的上皮效应  

We describe a protocol detailing the isolation of murine colonic crypts for the development of 3-dimensional colonoids. The established colonoids can then be terminally differentiated to reflect the cellular composition of the host epithelium prior to receiving an inflammatory challenge or being directed to establish an epithelial monolayer.

研究体 外 肠道炎症对已建立的小鼠结肠样的上皮影响

The intestinal epithelium plays an essential role in human health, providing a barrier between the host and the external environment. This highly dynamic ...