Name: in vitro and in vivo approaches to determine intestinal epithelial cell permeability
Uploaded: 2018-10-19T12:30:00
Description: Watch this Scientific Journal Video about In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability at JoVE.com

我们感谢哈佛医学院的杨百翰和女子医院的杰罗尔德博士, 他对完成这项研究的慷慨帮助。这项工作由中国国家自然科学基金 (赠款号81470804、31401229和 81200620)、江苏省自然科学基金 (赠款编号 BK20180838 和 BK20140319) 支持, 研究创新计划江苏省大学毕业生 (赠款编号 KYLX16-0116)、东吴大学高级研究项目 (赠款编号 SDY2015_06)、克罗恩 & 结肠炎基金会研究奖学金 (赠款号 310801)。

本研究获得了苏州大学剑桥-苏达基因组资源中心 (CAM) 动物护理与使用协议的批准。
1. Caco-2bbe 在多孔聚碳酸酯膜上的电镀和维护
<ol><li>在带有培养基的 T75 烧瓶中生长细胞 (DMEM 含有 10% FBS)。烧瓶应定期喂养, 具体取决于细胞密度。 注意: 为了获得最佳电镀, 细胞应迅速分裂, 并具有扁平的 "煎蛋" 形状, 这表明细胞处于生长阶段。</li>
	<li>一旦细胞80% 汇合, 从孵化器中取出烧?...

<ol>
	<li>Turner, 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=Intestinal+mucosal+barrier+function+in+health+and+disease.">Intestinal mucosal barrier function in health and disease.</a> Nature reviews. Immunology. 9, 799-809 (2009).</li><li>Odenwald, M. A., Turner, 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=The+intestinal+epithelial+barrier:+a+therapeutic+target?.">The intestinal epithelial barrier: a therapeutic target?.</a> Nature reviews. Gastroenterology &amp; hepatology. 14, 9-21 (2017).</li><li>Clarke, H., Soler, A. P., Mullin, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+kinase+C+activation+leads+to+dephosphorylation+of+occludin+and+tight+junction+permeability+increase+in+LLC-PK1+epithelial+cell+sheets.">Protein kinase C activation leads to dephosphorylation of occludin and tight junction permeability increase in LLC-PK1 epithelial cell sheets.</a> J. Cell Sci. 113 (Pt 18), 3187-3196 (2000).</li><li>Clayburgh, D. R., 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=A+differentiation-dependent+splice+variant+of+myosin+light+chain+kinase,+MLCK1,+regulates+epithelial+tight+junction+permeability.">A differentiation-dependent splice variant of myosin light chain kinase, MLCK1, regulates epithelial tight junction permeability.</a> J.Biol. Chem. 279, 55506-55513 (2004).</li><li>Su, L., 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=TNFR2+activates+MLCK-dependent+tight+junction+dysregulation+to+cause+apoptosis-mediated+barrier+loss+and+experimental+colitis.">TNFR2 activates MLCK-dependent tight junction dysregulation to cause apoptosis-mediated barrier loss and experimental colitis.</a> Gastroenterology. 145, 407-415 (2013).</li><li>Su, L., 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=Targeted+epithelial+tight+junction+dysfunction+causes+immune+activation+and+contributes+to+development+of+experimental+colitis.">Targeted epithelial tight junction dysfunction causes immune activation and contributes to development of experimental colitis.</a> Gastroenterology. 136, 551-563 (2009).</li><li>Zha, J. M., 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=Characterization+of+isoform+expression+and+subcellular+distribution+of+MYPT1+in+intestinal+epithelial+cells.">Characterization of isoform expression and subcellular distribution of MYPT1 in intestinal epithelial cells.</a> Gene. 588, 1-6 (2016).</li><li>He, W. Q., 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=Altered+contractile+phenotypes+of+intestinal+smooth+muscle+in+mice+deficient+in+myosin+phosphatase+target+subunit+1.">Altered contractile phenotypes of intestinal smooth muscle in mice deficient in myosin phosphatase target subunit 1.</a> Gastroenterology. 144, e1451-e1455 (2013).</li><li>He, W. Q., 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=Myosin+light+chain+kinase+is+central+to+smooth+muscle+contraction+and+required+for+gastrointestinal+motility+in+mice.">Myosin light chain kinase is central to smooth muscle contraction and required for gastrointestinal motility in mice.</a> Gastroenterology. 135, 610-620 (2008).</li><li>Li, H. S., 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=Myosin+regulatory+light+chain+phosphorylation+is+associated+with+leiomyosarcoma+development.">Myosin regulatory light chain phosphorylation is associated with leiomyosarcoma development.</a> Biomed. Pharmacother. 92, 810-818 (2017).</li><li>Clayburgh, D. R., 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=Epithelial+myosin+light+chain+kinase-dependent+barrier+dysfunction+mediates+T+cell+activation-induced+diarrhea+in+vivo.">Epithelial myosin light chain kinase-dependent barrier dysfunction mediates T cell activation-induced diarrhea in vivo.</a> J. Clin. Invest. 115, 2702-2715 (2005).</li><li>Wang, F., 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=Interferon-gamma+and+tumor+necrosis+factor-alpha+synergize+to+induce+intestinal+epithelial+barrier+dysfunction+by+up-regulating+myosin+light+chain+kinase+expression.">Interferon-gamma and tumor necrosis factor-alpha synergize to induce intestinal epithelial barrier dysfunction by up-regulating myosin light chain kinase expression.</a> Am. J. Pathol. 166, 409-419 (2005).</li><li>Ye, D., Ma, T. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+and+molecular+mechanisms+that+mediate+basal+and+tumour+necrosis+factor-alpha-induced+regulation+of+myosin+light+chain+kinase+gene+activity.">Cellular and molecular mechanisms that mediate basal and tumour necrosis factor-alpha-induced regulation of myosin light chain kinase gene activity.</a> J. Cell Mol. Med. 12, 1331-1346 (2008).</li><li>Turner, J. R., Buschmann, M. M., Romero-Calvo, I., Sailer, A., Shen, L. <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+molecular+remodeling+in+differential+regulation+of+tight+junction+300+permeability.">The role of molecular remodeling in differential regulation of tight junction 300 permeability.</a> Semin. Cell Dev. Biol. 36, 204-212 (2014).</li><li>Srinivasan, B., 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=TEER+measurement+techniques+for+in+vitro+barrier+model+systems.">TEER measurement techniques for in vitro barrier model systems.</a> J. Lab. Autom. 20 (2), 107-126 (2015).</li><li>Wang, F., 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=IFN-gamma-induced+TNFR2+expression+is+required+for+TNF-dependent+intestinal+epithelial+barrier+dysfunction.">IFN-gamma-induced TNFR2 expression is required for TNF-dependent intestinal epithelial barrier dysfunction.</a> Gastroenterology. 131, 1153-1163 (2006).</li><li>Peterson, M. D., Mooseker, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+enterocyte-like+brush+border+cytoskeleton+of+the+C2BBe+clones+of+the+human+intestinal+cell+line+Caco-2.">Characterization of the enterocyte-like brush border cytoskeleton of the C2BBe clones of the human intestinal cell line Caco-2.</a> J. Cell Sci. 102 (Pt 3), 581-600 (1992).</li><li>Wang, L., 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=Methods+to+determine+intestinal+permeability+and+bacterial+translocation+during+liver+disease.">Methods to determine intestinal permeability and bacterial translocation during liver disease.</a> J. Immunol. Methods. 421, 44-53 (2015).</li></ol>

协议中有几个关键步骤。Caco-2BBe (画笔边框表示) 细胞始终用于测量, 从 Caco-2 细胞系中选择, 用于表达画笔边界蛋白。Caco-2BBe 细胞有一个绒毛吸收表型时完全分化 (经过大约3周的文化后汇合)17。在测量过程中, 必须避免污染, 并对电极进行灭菌。由于该过程是非无菌的, 在大多数情况下, 测量只能在8小时内执行。在测量过程中, 我们确定了刀片内外两点的电阻值。不同部位的电阻测...

肠道上皮细胞不仅负责养分的吸收, 而且是抵御致病微生物和微生物毒素的重要屏障。此肠屏障功能由紧密的结通透性和上皮细胞完整性1、2、3和上皮屏障功能功能障碍与炎症性肠相关调节疾病 (IBD)。perijunctional 肌动球蛋白环 (PAMR) 位于与紧密交汇点紧密相连的细胞内。PAMR 的收缩是由肌球蛋白光链 (MLC) 调节的, 对于紧密接合渗透率4、5、6、7、8的调节至关重要. 9,10。肿瘤坏死因子 (TNF) 是上调肠上皮 MLC 激酶 (拿) 表达和诱导 occludin 内化11、12、13的肠道屏障丢失的中枢。
离子, 如 Na+和 Cl-可以通过孔隙或泄漏通路14跨越旁细胞空间。在 "漏" 上皮中, 三的变化主要反映改变的紧密结通透性。测量是一种常用的电生理学方法, 用于根据细胞膜的阻抗来量化紧密结通透性, 主要是 Na+和 Cl。不同的细胞类型, 包括肠道上皮细胞, 肺上皮细胞和血管内皮细胞, 已经报告了三的测量。这种方法的优点是, 测量是非侵入性的, 可用于实时监测活细胞。此外, 该方法对药物毒性研究15有参考价值。
Caco-2BBe 细胞是人上皮性大肠腺癌细胞, 其结构和功能类似于分化的小肠上皮细胞: 例如, 这些细胞有微绒毛和与小肠刷相关的酶边境。因此, 培养的 Caco-2BBe 单层膜被用作检测屏障功能的体外模型。
在小鼠中, 研究肠道旁细胞通透性的一种方法是通过测量 FITC-葡聚糖从腔内向血液中交叉的能力。因此, 肠道通透性可以通过 gavaging FITC-葡聚糖直接对小鼠进行评估, 并测量血液中的荧光。下面的协议描述了两种简单的方法来评估小肠上皮通透性的体外和体内。

<table>
	<tbody>
		<tr>
			<td>22G gavage needle</td>
			<td>VWR</td>
			<td>20068-608</td>
			<td></td>
		</tr>
		<tr>
			<td>4 kDa FITC-dextran</td>
			<td>Sigma</td>
			<td>46944</td>
			<td></td>
		</tr>
		<tr>
			<td>Avertin</td>
			<td>Sigma</td>
			<td>T48402</td>
			<td></td>
		</tr>
		<tr>
			<td>Black 96-well plates for fluorescence</td>
			<td>Fisher</td>
			<td>14-245-197A</td>
			<td></td>
		</tr>
		<tr>
			<td>C57/B6 mice</td>
			<td>Nanjing Biomedical Research Institute of Nanjing University</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Caco-2BBe cells</td>
			<td>ATCC</td>
			<td>CRL-2102</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextran sulphate sodium</td>
			<td>MP Biomedicals</td>
			<td>2160110</td>
			<td></td>
		</tr>
		<tr>
			<td>DMED with high glucose and sodium pyruvate&#160;</td>
			<td>Hyclone</td>
			<td>SH30243.01B</td>
			<td></td>
		</tr>
		<tr>
			<td>Epithelial (Volt/Ohm) Meter&#160;</td>
			<td>Millicell-ERS</td>
			<td>MERS00002</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Sinopharm ChemicalReagent</td>
			<td>10009218</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon tube (15 mL)</td>
			<td>Corning</td>
			<td>430791</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Gibco</td>
			<td>10437-028</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence microscope</td>
			<td>Olympus&#160;</td>
			<td>FV1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorometer</td>
			<td>Biotek</td>
			<td>Synergy 2</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td></td>
			<td></td>
			<td>138 mM NaCl, 0.3 mM Na2HPO4, 0.4 mM MgSO4, 0.5 mM MgCl2, 5.0 mM KCl, 0.3 mM KH2PO4, 15.0 mM HEPES, 1.3 mM CaCl2, 25 mM glucose&#160;</td>
		</tr>
		<tr>
			<td>IFNg</td>
			<td>PeproTech</td>
			<td>315-05-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Modular Tissue Embedding Center&#160;</td>
			<td>Leica</td>
			<td>EG1150H</td>
			<td></td>
		</tr>
		<tr>
			<td>Serum collection tubes</td>
			<td>Sarstedt</td>
			<td>41.1378.005</td>
			<td></td>
		</tr>
		<tr>
			<td>T75 flask&#160;</td>
			<td>corning</td>
			<td>430641</td>
			<td></td>
		</tr>
		<tr>
			<td>TNF</td>
			<td>PeproTech</td>
			<td>315-01A</td>
			<td></td>
		</tr>
		<tr>
			<td>Parraffin</td>
			<td>Sigma</td>
			<td>A6330-1CS</td>
			<td></td>
		</tr>
		<tr>
			<td>Polycarbonate membranes (Transwell)</td>
			<td>Costar</td>
			<td>3413</td>
			<td></td>
		</tr>
		<tr>
			<td>Pressure pump</td>
			<td>AUTOSCIENCE</td>
			<td>AP-9925</td>
			<td></td>
		</tr>
		<tr>
			<td>Rotary Microtomy</td>
			<td>Leica</td>
			<td>RM2235</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Gibco</td>
			<td>25200-056</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>Sinopharm ChemicalReagent</td>
			<td>10023418</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro and in vivo approaches to determine intestinal epithelial cell permeability

肠道屏障抵御致病微生物和微生物毒素。其功能由紧密的结通透性和上皮细胞完整性调节, 而肠道屏障功能的破坏有助于胃肠道和系统性疾病的发展。本文介绍了两种简单的方法来测量小肠上皮的通透性。在体外, Caco-2BBe 细胞被镀在组织培养井作为单层和测定电阻 (三) 可以用上皮 (伏/欧姆) 计测量。这种方法是令人信服的, 因为它的用户友好的操作和重复性。在体内,小鼠孕期 4 kDa 荧光素异硫氰酸酯 (FITC)-葡聚糖, 在采集的小鼠血清样本中测定 FITC-葡聚糖浓度, 以确定上皮通透性。口服灌胃提供准确剂量, 因此是测定体内肠道通透性的首选方法。这两种方法可以在体外和体内测量小肠上皮的通透性, 从而用于研究疾病与屏障功能的关系。

在培养中, Caco-2BBe 细胞作为单层生长, 慢慢分化为具有画笔边界的成熟吸收 enterocytes。在本协议中, Caco-2BBe 细胞在聚碳酸酯膜上镀上高密度, 细胞在播种后一天达到100% 融合。然而, 细胞在这个阶段是未分化的: 要完全区分细胞, 介质每2-3 天更换3周。细胞被细胞核和 F 肌动蛋白染色, 以显示未分化和分化细胞之间的差异。应力纤维在未分化细胞中明显可见。与未分化细胞相比...

Watch this Scientific Journal Video about In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability at JoVE.com

In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability

本文提出了两种方法来确定肠道屏障功能。在组织培养井中, 使用上皮表 (伏特/欧姆) 直接测量培养的上皮细胞的测定电阻。在小鼠中, 采用 FITC-葡聚糖灌胃法测定体内肠道通透性。

肠上皮细胞通透性的体外及体内测定方法

The intestinal barrier defends against pathogenic microorganism and microbial toxin. Its function is regulated by tight junction permeability and ...

本实验的总体目标是通过测量体外和体内肠道上皮细胞渗透性，研究上皮屏障功能障碍功能引起的疾病。此方法可以帮助回答肠道屏障功能领域的关键问题，例如与炎症性肠病相关的问题。该技术的主要优点是，肠道上皮细胞的渗透性可以在体外和体内进行评估。这种技术的影响延伸到胃肠道疾病的治疗或诊断，因为肠道上皮屏障功能障碍功能障碍有助于这些疾病。虽然这种方法可以提供对肠道屏障功能的洞察，它也可以应用于其他系统，如药物毒性研究和其他细胞类型的屏障完整性。此方法的可视化演示至关重要，因为某些步骤很难学习。现在可以通过文本描述来理解此方法的准确操作。首先，在具有中等的T75烧瓶中生长Caco-2bbe细胞。根据细胞密度定期喂料瓶。当细胞是80%汇合时，取出培养基，使用1-2毫升无菌PBS，无需钙冲洗。加入1.5毫升的三辛-EDTA，轻轻摇动烧瓶。然后，在37摄氏度的培养箱中放置20分钟，不摇摆。当细胞进行复数时，将含有多孔聚碳酸酯膜的插入物放入24个孔板中。然后将1毫升培养基加入基底室，基底室是膜的下部空间。在烧瓶中加入5毫升的培养，用力将细胞对瓶内移液5至10次，将培养成松散的单个细胞，或两到三个细胞团。板 0.166 毫升的细胞进入膜的上部空间的腔室。然后在37摄氏度下孵育板长达三周。每周三次给细胞喂气，使用压力泵从每一个井的基底室小心地吸气介质。然后轻轻地将一毫升介质滴入每个刀片的腔室。对于细胞因子研究，在跨皮电阻或TER测量前一天，用介质替换基底介质，每毫升干扰素伽马含有10毫微克。在实验当天，用每毫升TNF含有2.5或7.5毫微克的HSSS代替介质。要校正仪表，请将校正电极插入输入端口并选择欧姆模式。然后使用螺丝刀调整 R 调整螺钉，直到仪表显示读数为 1，000 欧姆。在 70%乙醇中对电极进行消毒 15 至 30 分钟，让电极风干 15 秒。然后在实验细胞培养基中冲洗电极。接下来，打开电源并选择欧姆模式。在保持电极垂直的同时，小心地将电极桥的长端放入基底室，确保它们接触碟子。然后将短端放入腔室，确保它们位于介质表面之下，但位于组织培养物插入物之上。在细胞因子治疗后 0、1、2、3 和 4 小时测量样品和空白刀片的电阻。然后记录阻力。要实现不同板格式的一致性，请计算电阻的产品和有效膜面积，如下所示。对于 24 孔插入，有效膜面积为 0.33 平方厘米。要诱发结肠炎，将 DSS 添加到自 <3>化水中，最终浓度为每体积 3.5% 的重量。然后使用 DSS 溶液替换 8 周大雄性 C57 黑六只小鼠的笼子里的饮用水，总共 7 天。给常规饮用水没有 DSS 控制小鼠。第七天后，将 DSS 水切换到常规饮用水。每天，体重小鼠，并评估根据疾病严重程度定义的临床分数四个参数：直肠脱垂，大便一致性，出血和活动。从这些参数中总结分数，以最终的临床评分。分析DSS治疗后7天结肠组织病理学状况，根据文本协议对小鼠实施安乐死后，分离结肠和结肠，测量结肠的长度。从远端结肠中切下0.5厘米的段，将组织固定在含有10毫升10%甲醛的15毫升猎鹰管中。用分级乙醇和二甲苯清洗固定组织。然后将组织嵌入石蜡中，并切割六毫米部分，用于血氧素和 eosin 染色。为了测量DSS诱导小鼠的上皮屏障渗透性，在DSS给给开始7天后，对小鼠禁食3小时。通过解套针，确保无菌，然后在无菌水中使用150微升每毫升80毫克，4千达吨 FITC dextran 对小鼠进行加气，并保留未使用的 FITC dextran，以测量血清采集后的标准曲线。接下来，对鼠标进行加权以进行渗透性计算。四个小时后，通过IP注射麻醉小鼠后，通过缺乏反射和胡须运动确认适当的麻醉。然后把老鼠放在热块上五分钟。用一把剪刀将一厘米的尾巴夹住，从尾巴收集100微升血液，放入血清收集管中。在10，000倍g和室温下旋转采集的血液10分钟。用水，稀释血清一到四。然后，要制定一个标准曲线，使用水来准备未使用的 FITC dextran 的稀释。将血清和标准曲线样品添加到 96 个孔板中，每孔添加 100 微升。使用板读卡器，读取 485 和 528 发射时荧光。基于标准曲线计算渗透率值，并乘以四以校正稀释。将 FITC dextran 的浓度除以权重以使值正常化。这有助于使如果小鼠生病并且体重减轻，FITC dextran 分娩的差异正常化。此处显示的 Caco-2bbe 细胞标有核和 F-actin 染色，以显示未分化和分化细胞之间的差异。应力纤维在未分化的细胞中清晰可见。分化细胞的体积较小，核更大，应力纤维比未分化的细胞少。TNF 是通过 MLCK 依赖紧密结调节对肠道屏障损失的核心。如图所示，TNF 以剂量依赖的方式显著降低 Caco-2bbe 单层 TER，这表明 TNF 增加了上皮渗透性。与最初的体重相比，使用 DSS 治疗的小鼠体重减轻很大。结肠炎的严重程度由直肠脱垂、大便一致性、出血和活动来评分。沾染血氧素和 eosin 的结肠组织的横截面显示 DSS 治疗小鼠的结肠粘膜损伤。此外，在经过 DSS 治疗的小鼠中，结肠墓穴的长度以及结肠本身的长度也减少了。最后，与对照小鼠相比，经过DSS治疗的小鼠的 FITC dextran 染色水平大约增加了两倍。在尝试此过程时，重要的是要记住通过标准化操作过程和利用统计方法来尽量减少错误。该技术开发后，为肠道屏障功能领域的研究人员探索模型生物体和细胞系中的胃肠道疾病铺平了道路。看完这段视频后，您应该对如何通过测量 TER、Gavaging FITC dextran 和观察形态和组体化学变化来确定肠道上皮渗透性有了很好的了解。

Research

JoVE Journal

Biology

In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability

Visualization of MG53-mediated Cell Membrane Repair Using in vivo and in vitro Systems

Visualization of MG53-mediated Cell Membrane Repair Using in vivo and in vitro Systems

MG53介导的细胞膜使用维修的可视化在体内在体外系统

Repair of acute injury to the cell membrane is an elemental process of normal cellular physiology, and defective membrane repair has been linked to many ...

科研

生物学

Chromatin Isolation by RNA Purification (ChIRP)

Chromatin Isolation by RNA Purification ChIRP

RNA纯化的染色质隔离（CHIRP）

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

核糖体束缚新生多肽的分离<em&gt;在体外</em&gt;确定沿mRNA的翻译暂停网站

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration

在毛发再生的上皮 - 间充质信号的快速遗传分析

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an ...

Real Time Analysis of Metabolic Profile in Ex Vivo Mouse Intestinal Crypt Organoid Cultures

Real Time Analysis of Metabolic Profile in Ex Vivo Mouse Intestinal Crypt Organoid Cultures

代谢谱的实时分析<em&gt;离体</em&gt;鼠标肠隐窝类器官培养

The small intestinal mucosa exhibits a repetitive architecture organized into two fundamental structures: villi, projecting into the intestinal lumen and ...

Genetic and Biochemical Approaches for In Vivo and In Vitro Assessment of Protein Oligomerization: The Ryanodine Receptor Case Study

Genetic and Biochemical Approaches for In Vivo and In Vitro Assessment of Protein Oligomerization: The Ryanodine Receptor Case Study

遗传和生物化学方法<em&gt;在体内</em&gt;和<em&gt;体外</em&gt;蛋白齐聚评估：兰尼碱受体案例研究

Oligomerization is often a structural requirement for proteins to accomplish their specific cellular function. For instance, tetramerization of the ...

Glomerular Outgrowth as an Ex Vivo Assay to Analyze Pathways Involved in Parietal Epithelial Cell Activation

球状外生长作为前维沃分析分析涉及的路径的表皮上皮细胞激活

Parietal epithelial cell (PEC) activation is one of the key factors involved in the development and progression of glomerulosclerosis. Inhibition of ...

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

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

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 ...

In Vivo Vascular Permeability Detection in Mouse Submandibular Gland

In Vivo Vascular Permeability Detection in Mouse Submandibular Gland

体内 小鼠下颌下腺血管通透性检测

Saliva plays an important role in oral and overall health. The intact endothelial barrier function of blood vessels enables saliva secretion, whereas the ...