Name: a novel method for the culture and polarized stimulation of human intestinal mucosa explants
Uploaded: 2013-05-01T12:00:00
Description: Watch this Scientific Journal Video about A Novel Method for the Culture and Polarized Stimulation of Human Intestinal Mucosa Explants at JoVE.com

我们感谢埃里卡Mileti，优异的技术支持和安东尼奥·SABATINO博士提供氧舱。 资金来源 ：本工作得到补助从欧盟第七框架计划（IBDase ERC：Dendroworld），和基金会Cariplo的MR和支持居里夫人国际培训移动网络（相声，赠款协议编号：21553-2） KT。

1。获取和组织准备<ol><li>组织（健康或粘膜IBD）是在手术过程中获得。一旦切除试样转移到实验室尽快，保持它的Ca + + / Mg的+ +的HBSS缓冲液在4℃或冰面上青/链霉素补充。 </li></ol> *试样的大小取决于可用的组织得到的组织的一部分，它是严格诊断是没有必要的，并且可以相差很大健康IBD主体之间。健康的组织切除患者结肠癌手术。 <ol start="2"><li>轻轻洗净的?...

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	<li>Schuller, S., Lucas, M., Kaper, J. B., Giron, J. A., Phillips, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ex+vivo+response+of+human+intestinal+mucosa+to+enteropathogenic+Escherichia+coli+infection.">The ex vivo response of human intestinal mucosa to enteropathogenic Escherichia coli infection.</a> Cell. Microbiol. 11, 521-530 (2009).</li><li>Tsilingiri, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probiotic+and+postbiotic+activity+in+health+and+disease:+comparison+on+a+novel+polarised+ex-vivo+organ+culture+model.">Probiotic and postbiotic activity in health and disease: comparison on a novel polarised ex-vivo organ culture model.</a> Gut. , (2012).</li><li>Tsilingiri, K., Rescigno, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Should+probiotics+be+tested+on+ex+vivo+organ+culture+models?.">Should probiotics be tested on ex vivo organ culture models?.</a> Gut Microbes. , (2012).</li><li>Mileti, E., Matteoli, G., Iliev, I. D., Rescigno, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+the+immunomodulatory+properties+of+three+probiotic+strains+of+Lactobacilli+using+complex+culture+systems:+prediction+for+in+vivo+efficacy.">Comparison of the immunomodulatory properties of three probiotic strains of Lactobacilli using complex culture systems: prediction for in vivo efficacy.</a> PLoS One. 4, e7056 (2009).</li><li>Cencic, A., Langerholc, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+cell+models+of+the+gut+and+their+applications+in+food+microbiology--a+review.">Functional cell models of the gut and their applications in food microbiology--a review.</a> Int. J. Food Microbiol. 141, S4-S14 (2010).</li><li>te Velde, A. A., Verstege, M. A., Hommes, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Critical+appraisal+of+the+current+practice+in+murine+TNBS-induced+colitis.">Critical appraisal of the current practice in murine TNBS-induced colitis.</a> Inflamm. Bowel Dis. 12, 995-999 (2006).</li><li>Senior, P. V., Pritchett, C. J., Sunter, J. P., Appleton, D. R., Watson, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crypt+regeneration+in+adult+human+colonic+mucosa+during+prolonged+organ+culture.">Crypt regeneration in adult human colonic mucosa during prolonged organ culture.</a> J. Anat. 134, 459-469 (1982).</li><li>Browning, T. H., Trier, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organ+culture+of+mucosal+biopsies+of+human+small+intestine.">Organ culture of mucosal biopsies of human small intestine.</a> J. Clin. Invest. 48, 1423-1432 (1969).</li><li>Besselink, M. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probiotic+prophylaxis+in+predicted+severe+acute+pancreatitis:+a+randomised,+double-blind,+placebo-controlled+trial.">Probiotic prophylaxis in predicted severe acute pancreatitis: a randomised, double-blind, placebo-controlled trial.</a> Lancet. 371, 651-659 (2008).</li><li>Lakhdari, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+NF-kappaB+modulation+capabilities+within+human+intestinal+commensal+bacteria.">Identification of NF-kappaB modulation capabilities within human intestinal commensal bacteria.</a> J. Biomed. Biotechnol. 2011, 282356 (2011).</li></ol>

需要有关生理模型人体肠道黏膜上的治疗方法，可以有效地测试一直很重视。许多研究人员已经发现在两种细胞和小鼠模型6迄今所使用为该目的而潜在的工件和缺陷。事实上，即使有前途的临床前数据往往是获得，这些很少转化为显着的临床益处。 在这项工作中所描述的方法，提出了一个相对简单的，但有效的，新颖的适应人体肠道粘膜偏振方式的外植体的培养和?...

<table border="1">
	<tbody>
		<tr>
			<td>Regeant</td>
			<td>Company</td>
			<td>Catalog No.</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Reagents</td>
		</tr>
		<tr>
			<td>10X HBSS</td>
			<td>Eurocalne</td>
			<td>ECM4006XL</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Peni/Strepto</td>
			<td>Lonza</td>
			<td>DE17602E</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Gentamycin</td>
			<td>Gibco</td>
			<td>15750-037</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Lonza</td>
			<td>BE12614</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>FBS-Na</td>
			<td>Gibco</td>
			<td>16000-044</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>100X ITS-X</td>
			<td>Gibco</td>
			<td>51500-056</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>Tebu-bio</td>
			<td>100-15</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Lonza</td>
			<td>BE17605E</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Non essential aminoacids 100X</td>
			<td>Gibco</td>
			<td>11140-035</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NaPyr</td>
			<td>Gibco</td>
			<td>11360-039</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Materials</td>
		</tr>
		<tr>
			<td>Cloning cylinders 6x8 mm</td>
			<td>BellCo</td>
			<td>2090-00608</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cloning cylinders 8x8 mm</td>
			<td>BellCo</td>
			<td>2090-0080</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cloning cylinders 10x10 mm</td>
			<td>BellCo</td>
			<td>2090-01010</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Metal grids</td>
			<td>Home made</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Centre well organ culture plate</td>
			<td>BD Falcon</td>
			<td>353037</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Surgical glue (Vetbond)</td>
			<td>3M</td>
			<td>1469SB</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Oxygen Jars</td>
			<td>Home made</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Oxygen tanks</td>
			<td>Air Liquid Sanit&agrave;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

a novel method for the culture and polarized stimulation of human intestinal mucosa explants

目前很少有车型存在，逼真地模拟复杂的人类肠道内的微环境，在那里发生的各种相互作用。适当的稳态直接取决于这些相互作​​用的，因为它们形成整个的免疫反应对食物抗原诱导免疫耐受，而在同一时间对意外地与食物摄入的病原微生物的有效的免疫反应。 肠道动态平衡也被保留通过各种微生物（包括食品相关的有益的细菌菌株）和主机，调节附件/降解粘液之间复杂的相互作用，生产抗菌肽上皮屏障，而“教育”上皮细胞表型独特，肠道居民淋巴细胞人口控制的耐受性或免疫原性。这些相互作用到目前为止已经很难复制的体外实验可以使用培养的细胞系或外周血单核细胞。此外，小鼠模型显着不同的元件相对于人体肠道对肠粘膜（粘液层的组织，共生细菌群落）。因此在诊所被带到重要的压力相关的或病理情况下，如肠易激综合征，炎性肠病或结直肠癌的治疗方法有多种，研究一直难以开展。 为了解决这些问题，我们开发了一种新的系统，使我们能够刺激人体肠道黏膜植保留其在原位空调主机菌群和免疫反应，极化方式。偏振光根尖刺激引起的免疫反应的结果，非常重要的。已经一再证明，相同的刺激，可以产生完全不同的反应，当他们绕过心尖面对肠道外延thelium basolaterally或刺激上皮细胞进入固有层组件的直接接触，切换从耐受性表型的免疫原性，并在区域造成不必要的过度的炎症反应。 通过胶粘的分隔的区域的顶端面的粘膜刺激对将要描述在协议中的一个洞穴缸实现极化刺激。我们用这个模型来考察，其中包括三个不同的乳酸杆菌菌株的不同影响。我们发现，这个模型系统评估益生菌的免疫调节特性，在健康和疾病的情况下是非常强大的。

我们成功地保持健康和传染性法氏囊病的人类文化肠粘膜至少24小时保护组织的福祉。根据先前的观察1，这是唯一可能的，如果培养时间（至少85％的总）的大多数发生在O 2，组织没有生存24小时，在传统的孵化器（ 图2）。我们也显示出，取决于上的的刺激（心尖比基底侧）和刺激的免疫原性2,3的极性在此模型上可观察到的外植体的不同的反应。 <p class="jo...

Watch this Scientific Journal Video about A Novel Method for the Culture and Polarized Stimulation of Human Intestinal Mucosa Explants at JoVE.com

A Novel Method for the Culture and Polarized Stimulation of Human Intestinal Mucosa Explants

我们引入了一种新的方法，为维护人类文化和监测至少24小时应对各类刺激肠黏膜。有了我们的方法中，维持组织的极性，可用于生理刺激通过心尖路由。

文化与偏振刺激人体肠道黏膜植的新方法

Few models currently exist to realistically simulate the complex human intestine's micro-environment, where a variety of interactions take place. Proper ...

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