この作品は、カナダ衛生研究所からの補助金（CIHR）およびクローン病とカナダの大腸炎財団（CCFC）によってサポートされています。

<ol>
	<li>Sands, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+symptom+to+diagnosis:+clinical+distinctions+among+various+forms+of+intestinal+inflammation.">From symptom to diagnosis: clinical distinctions among various forms of intestinal inflammation.</a> Gastroenterology. 126, 1518-1532 (2004).</li><li>Danese, S., Fiocchi, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Etiopathogenesis+of+inflammatory+bowel+diseases.">Etiopathogenesis of inflammatory bowel diseases.</a> World J. Gastroenterol. 12, 4807-4812 (2006).</li><li>Wirtz, S., Neufert, C., Weigmann, B., Neurath, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemically+induced+mouse+models+of+intestinal+inflammation.">Chemically induced mouse models of intestinal inflammation.</a> Nat. Protoc. 2, 541-546 (2007).</li><li>Axelsson, L. G., Landstrom, E., Goldschmidt, T. J., Gronberg, A., Bylund-Fellenius, A. C. <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+(DSS)+induced+experimental+colitis+in+immunodeficient+mice:+effects+in+CD4(+)-cell+depleted,+athymic+and+NK-cell+depleted+SCID+mice.">Dextran sulfate sodium (DSS) induced experimental colitis in immunodeficient mice: effects in CD4(+)-cell depleted, athymic and NK-cell depleted SCID mice.</a> Inflamm. Res. 45, 181-191 (1996).</li><li>Egger, B., Bajaj-Elliott, M., MacDonald, T. T., Inglin, R., Eysselein, V. E., Büchler, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterisation+of+acute+murine+dextran+sodium+sulphate+colitis:+cytokine+profile+and+dose+dependency.">Characterisation of acute murine dextran sodium sulphate colitis: cytokine profile and dose dependency.</a> Digestion. 62, 240-248 (2000).</li><li>Krawisz, J. E., Sharon, P., Stenson, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+assay+for+acute+intestinal+inflammation+based+on+myeloperoxidase+activity.+Assessment+of+inflammation+in+rat+and+hamster+models.">Quantitative assay for acute intestinal inflammation based on myeloperoxidase activity. Assessment of inflammation in rat and hamster models.</a> Gastroenterology. 87, 1344-1350 (1984).</li><li>Cooper, H. S., Murthy, S. N., Shah, R. S., Sedergran, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinicopathologic+study+of+dextran+sulfate+sodium+experimental+murine+colitis.">Clinicopathologic study of dextran sulfate sodium experimental murine colitis.</a> Lab. Invest. 69, 238-249 (1993).</li><li>Smith, J. W., Castro, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relation+of+peroxidase+activity+in+gut+mucosa+to+inflammation.">Relation of peroxidase activity in gut mucosa to inflammation.</a> Am. J. Physiol. 234, R72-R79 (1978).</li><li>Ghia, J. E., Blennerhassett, P., Kumar-Ondiveeran, H., Verdu, E. F., Collins, S. 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>

利害の衝突は宣言されません。

DSS大腸炎は腸の炎症の広く使用されている化学的に誘発されたモデルです。このモデルでは、マウスは上皮細胞を消化管や粘膜関門の整合性を妨害するために有毒であると考えられているDSS、を添加した飲料水を与えられます。DSSの10の投与は、軟便、糞便出血、によって特徴づけられる急性大腸炎を誘発すると顆粒球と浸潤10 DSS投与中には、大腸炎は通常、便潜血検査で評...

<table border="1"><tbody><tr><td> 試薬/装置の名前： </td><td> 会社 </td><td> カタログ番号 </td><td> コメント </td></tr><tr><td>エッペンドルフセイフ - ロックのマイクロ遠心チューブ（液2ml） </td><td>エッペンドルフ</td><td> 0030 120.094 </td><td></td></tr><tr><td> BioTekのEL808吸光度プレートリーダー</td><td> BioTekの</td><td> EL808 </td><td></td></tr><tr><td>デキストラン硫酸ナトリウム塩の試薬グレード（分子量36,000-50,000ダ） </td><td> MPのバイオメディカル</td><td> 160110 </td><td></td></tr><tr><td> GEN5（ソフトウェア） </td><td> BioTekの</td><td>バージョン1.10.8 </td><td></td></tr><tr><td>臭化ヘキサデシルトリメチルアンモニウム（HTAB） </td><td>シグマアルドリッチ</td><td> H5882 - 100G </td><td></td></tr><tr><td>過酸化水素、水に30重量％溶液</td><td>シグマアルドリッチ</td><td> 216763 </td><td> 2〜8℃に保存</td></tr><tr><td>マイクロテストPL96ウェル平底を食べた</td><td>ザルスタット</td><td> 82.1581 </td><td> 1回限りの使用</td></tr><tr><td> o -ジアニシジン</td><td>シグマアルドリッチ</td><td> D - 3252 </td><td>敏感点灯。 2〜8℃に保存</td></tr><tr><td>リン酸カリウム、二塩基性</td><td>カレドン</td><td> 6620から1 </td><td></td></tr><tr><td>リン酸カリウム、一塩基</td><td> EMDケミカルズ</td><td> PX1565 - 1 </td><td></td></tr><tr><td>プロテアーゼ阻害剤カクテル</td><td>シグマアルドリッチ</td><td> P8340 </td><td> -20℃で保存</td></tr><tr><td>トリトンX - 100 </td><td>シグマアルドリッチ</td><td> T8787 </td><td></td></tr><tr><td>組織Lyser IIのための炭化タングステンビーズ</td><td> QUIAGEN </td><td> 69997 </td><td></td></tr></tbody></table>

investigating intestinal inflammation in dss-induced model of ibd

炎症性腸疾患（IBD）は腸の病態の範囲、潰瘍性大腸炎（UC）およびクローン病（CD）であるの最も一般的に網羅しています。 UCとCD、両方が大腸に存在する場合、下痢、直腸出血、腹痛、体重減少を含めることができます同じような症状のプロファイルを生成する。1 IBDの病因は不明であるが、それは両方を含む多因子疾患として記述されています遺伝と環境のコンポーネント2。 IBDのいくつかの機能に似ている結腸炎症の数々と可変の動物モデルがあります。デキストラン硫酸ナトリウム（DSS）などの大腸炎を誘発する化学物質の特定の濃度のものが必要な行政へのある種の感受性系統、で自発的に生じるものから大腸炎の範囲の動物モデル。腸の炎症の化学物質誘発性モデルは、IBDの最も一般的に使用されると最高に記載のモデルです。投与により飲料水におけるDSSの意気込みは、管理プロトコルに応じて、急性または慢性大腸炎を生じる。時には直腸出血の証拠で、DSS展示体重減少および軟便または下痢の症状を与えられた3匹の動物が。4,5ここで、我々はこれによってメソッドについて説明大腸炎の開発と、その結果炎症反応は、DSSの投与後に特徴づけることができる。これらのメソッドは、炎症の代理マーカーとして使用することができる組織学的ヘマトキシリン/エオシン染色コロンのセクションの解析、炎症性サイトカインの測定、およびミエロペルオキシダーゼの測定（MPO）活性を、6が含まれています病気の状態で炎症反応の程度は、臨床症状の有無によって、または粘膜組織の組織学の変化によって評価することができる。結腸の組織学的損傷は、陰窩構造、炎症性細胞浸潤、筋肉tの損失を考慮したスコアリングシステムを用いて評価されるhickening、杯細胞の枯渇、およびcrypt膿瘍。定量的に7、インターロイキン（IL）-1β、IL - 6、腫瘍壊死因子（TNF）-αなどの急性炎症性、、と炎症性サイトカインのレベルを用いて決定することができる従来のELISA法。さらに、MPO活性は比色アッセイを用いて測定することができ、炎症の指標として使用さ8。 実験的大腸炎では、疾患の重症度は、多くの場合、MPO活性の増加および炎症性サイトカインの高いレベルと相関している。大腸炎の重症度や炎症関連損傷をヘマトキシリン​​/エオシン染色結腸組織切片を用いて腸の病理組織学的状態を評価することに加えて、便の硬さと出血を調べることによって評価することができる。結腸組織の断片は、MPO活性とサイトカイン産生を決定するために使用することができます。一緒に、これらの措置は、腸内炎症反応を評価するために使用することができます。実験的大腸炎の動物モデル。

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

炎症性腸疾患の実験モデルは、私たちは病因に関連する複雑な自然免疫と獲得免疫応答を検討することを可能にした。組織学的スコアリング、炎症性サイトカインおよびミエロペルオキシダーゼ活性の定量化を使用して、一つは炎症性腸疾患に見られるこれらの応答を評価するために始めることができます。

IBDのDSS誘発モデルにおける腸炎の調査

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

66.8K ビュー.  McMaster University. 炎症性腸疾患の実験モデルは、私たちは病因に関連する複雑な自然免疫と獲得免疫応答を検討することを可能にした。組織学的スコアリング、炎症性サイトカインおよびミエロペルオキシダーゼ活性の定量化を使用して、一つは炎症性腸疾患に見られるこれらの応答を評価するために始めることができます。

ビデオ: Investigating Intestinal Inflammation in DSS-induced Model of IBD

Investigating the Microbial Community in the Termite Hindgut - Interview

Jared Leadbetter explains why the termite-gut microbial community is an excellent system for studying the complex interactions between microbes.     The symbiotic relationship existing between the host insect and lignocellulose-degrading gut microbes is explained, as well as the industrial uses of these microbes for degrading plant biomass and generating biofuels.

Jared Leadbetter explains why the termite-gut microbial community is an excellent system for studying the complex interactions between microbes. The symbiotic relationship existing between the host insect and lignocellulose-degrading gut microbes is explained, as well as the industrial uses of these microbes for degrading plant biomass and generating biofuels.

インタビュー - シロアリ後腸内微生物群集の調査

Jared Leadbetter explains why the termite-gut microbial community is an excellent system for studying the complex interactions between microbes.     The ...

生物学

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.

反芻動物の腸ループのカテーテル

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 ...

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.

ラット腸間膜具象化：血管新生に関与する細胞のダイナミクスを調べるためのモデル

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

Investigating the Spreading and Toxicity of Prion-like Proteins Using the Metazoan Model Organism C. elegans

Prions are unconventional self-propagating proteinaceous particles, devoid of any coding nucleic acid. These proteinaceous seeds serve as templates for the conversion and replication of their benign cellular isoform. Accumulating evidence suggests that many protein aggregates can act as self-propagating templates and corrupt the folding of cognate proteins. Although aggregates can be functional under certain circumstances, this process often leads to the disruption of the cellular protein homeostasis (proteostasis), eventually leading to devastating diseases such as Alzheimer&#8217;s disease (AD), Parkinson&#8217;s disease (PD), Amyotrophic lateral sclerosis (ALS), or transmissible spongiform encephalopathies (TSEs). The exact mechanisms of prion propagation and cell-to-cell spreading of protein aggregates are still subjects of intense investigation. To further this knowledge, recently a new metazoan model in Caenorhabditis elegans, for expression of the prion domain of the cytosolic yeast prion protein Sup35 has been established. This prion model offers several advantages, as it allows direct monitoring of the fluorescently tagged prion domain in living animals and ease of genetic approaches. Described here are methods to study prion-like behavior of protein aggregates and to identify modifiers of prion-induced toxicity using C. elegans.

Investigating the Spreading and Toxicity of Prion-like Proteins Using the Metazoan Model Organism C. elegans

Prion-like propagation of protein aggregates has recently emerged as being implicated in many neurodegenerative diseases. The goal of this protocol is to describe, how to use the nematode C. elegans as a model system to monitor protein spreading and to investigate prion-like phenomena.

拡散とプリオン様タンパク質の毒性は後生動物モデル生物を用いて調査<em&gt; C。エレガンス</em

Prions are unconventional self-propagating proteinaceous particles, devoid of any coding nucleic acid. These proteinaceous seeds serve as templates for ...

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.

レンチウイルス形質導入と腸オルガノイドの下流分析のためのプロトコル

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

Cryosectioning Method for Microdissection of Murine Colonic Mucosa

The colonic mucosal tissue provides a vital barrier to luminal antigens. This barrier is composed of a monolayer of simple columnar epithelial cells. The colonic epithelium is dynamically turned over and epithelial cells are generated in the stem cell containing crypts of Lieberk&#252;hn. Progenitor cells produced in the crypt-bases migrate toward the luminal surface, undergoing a process of cellular differentiation before being shed into the gut lumen. In order to study these processes at the molecular level, we have developed a simple method for the microdissection of two spatially distinct regions of the colonic mucosa; the proliferative crypt zone, and the differentiated surface epithelial cells. Our objective is to isolate specific crypt and surface epithelial cell populations from mouse colonic mucosa for the isolation of RNA and protein.

Here we describe a simple method for the isolation of spatially distinct murine colonic epithelial surface cells from the underlying crypt-base cells.

マウス結腸粘膜の顕微解剖のための凍結切片法

The colonic mucosal tissue provides a vital barrier to luminal antigens. This barrier is composed of a monolayer of simple columnar epithelial cells. The ...

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.

腸の部分的な閉塞のマウス モデル

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

The MUB40 Peptide for Use in Detecting Neutrophil-Mediated Inflammation Events

Here, we provide a protocol involving the use of MUB40, a synthesized peptide with the ability to bind glycosylated lactoferrin stored at high concentrations in specific and tertiary granules of neutrophils. This protocol details how MUB40&#160;conjugated directly to a fluorophore can be used to stain neutrophils in fixed/permeabilized tissues as well as how this can be used in live-cell imaging to assay for neutrophil activation and de-granulation. Neutrophil detection methods are limited to species-specific monoclonal antibodies, which are not always suitable for certain applications. MUB40 does not penetrate the cell membrane and is thus excluded from lactoferrin stored in non-activated/non-permeabilized neutrophils. MUB40 has the added benefit of recognizing lactoferrin from a broad host range, making it especially useful for comparing results in studies involving multiple research models, reducing the number of duplicate reagents, and simplifying protocols through single-step staining.

The MUB40 Peptide for Use in Detecting Neutrophil-Mediated Inflammation Events

Here, we present a protocol to detect the presence of neutrophils in fixed/permeabilized histology sections and assess the activation state of live purified neutrophils. In particular, the MUB40&#160;peptide binds lactoferrin present in neutrophil-specific and tertiary granules. Exposure of the granule contents through either permeabilization or neutrophil activation allows for the marking of neutrophils.

炎症の好中球を介したイベントの検出で使用するムバーラク40ペプチド

Here, we provide a protocol involving the use of MUB40, a synthesized peptide with the ability to bind glycosylated lactoferrin stored at high ...

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.

EYFP タグ付き CENP-A (EYFP-CENP-A) のユビキタス性を同定するための質量分析分析

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.

オルガノイド由来の上皮単分子膜:腸管バリア機能のための臨床的に関連するインビトロモデル

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

IBDのDSS誘発モデルにおける腸炎の調査

要約

さらに動画を探す

この動画の章

インタビュー - シロアリ後腸内微生物群集の調査

反芻動物の腸ループのカテーテル

ラット腸間膜具象化：血管新生に関与する細胞のダイナミクスを調べるためのモデル

拡散とプリオン様タンパク質の毒性は後生動物モデル生物を用いて調査

レンチウイルス形質導入と腸オルガノイドの下流分析のためのプロトコル

マウス結腸粘膜の顕微解剖のための凍結切片法

腸の部分的な閉塞のマウスモデル

炎症の好中球を介したイベントの検出で使用するムバーラク₄₀ペプチド

EYFP タグ付き CENP-A (EYFP-CENP-A) のユビキタス性を同定するための質量分析分析

オルガノイド由来の上皮単分子膜:腸管バリア機能のための臨床的に関連するインビトロモデル

IBDのDSS誘発モデルにおける腸炎の調査

要約

さらに動画を探す

この動画の章

インタビュー - シロアリ後腸内微生物群集の調査

反芻動物の腸ループのカテーテル

ラット腸間膜具象化：血管新生に関与する細胞のダイナミクスを調べるためのモデル

拡散とプリオン様タンパク質の毒性は後生動物モデル生物を用いて調査

レンチウイルス形質導入と腸オルガノイドの下流分析のためのプロトコル

マウス結腸粘膜の顕微解剖のための凍結切片法

腸の部分的な閉塞のマウス モデル

炎症の好中球を介したイベントの検出で使用するムバーラク40ペプチド

EYFP タグ付き CENP-A (EYFP-CENP-A) のユビキタス性を同定するための質量分析分析

オルガノイド由来の上皮単分子膜:腸管バリア機能のための臨床的に関連するインビトロモデル

腸の部分的な閉塞のマウスモデル

炎症の好中球を介したイベントの検出で使用するムバーラク₄₀ペプチド