Name: induction of intestinal inflammation by adoptive transfer of cbir1 tcr transgenic cd4+ t cells to immunodeficient mice
Uploaded: 2021-12-16T14:30:00
Description: Watch this Scientific Journal Video about Induction of Intestinal Inflammation by Adoptive Transfer of CBir1 TCR Transgenic CD4+ T Cells to Immunodeficient Mice at JoVE.com

这项工作得到了美国国立卫生研究院拨款DK125011，AI150210和DK124132，德克萨斯大学系统STAR奖（Y.C.）和德克萨斯大学加尔维斯顿分校（W.Y.）的James W. McLaughlin奖学金基金的部分支持。图 1 是使用 BioRender.com 创建的。

所有动物程序均根据德克萨斯大学医学分会的动物使用和护理委员会进行。CBir1 TCR Tg小鼠由阿拉巴马大学伯明翰分校的Charles Elson博士提供。CBir1 TCR Tg小鼠可以是雌性或雄性，但应该在8-12周。C57BL / 6背景上的 Rag1-/- 小鼠是从杰克逊实验室获得的10。 Rag1-/- 小鼠必须是性别和年龄匹配的，可以使用雄性或雌性，但应该在8-12周。整个协议总结如图 <strong c...

<ol>
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虽然每个步骤对于该结肠炎模型的可重复性都至关重要，但有几个关键步骤。受体Rag-/-小鼠应接受足够的活的naϊve CD4 + T细胞以诱导肠道炎症。我们使用脾脏来分离幼稚的CD4 + T细胞而不是MLN。因为MLNs中幼稚CD4 + T细胞的产量远低于脾脏。CD62L在幼稚T细胞中高度表达，CD44和CD25是T细胞的活化标志物13，14。在这项研究...

炎症性肠病（IBD），主要包括克罗恩病（CD）和溃疡性结肠炎（UC），其特征是胃肠道的慢性复发缓解炎症，影响全球数百万人1。IBD的发展和发病机制涉及几个因素，包括遗传易感性、肠道微生物群、免疫反应、饮食和生活方式2。然而，IBD的确切机制仍未完全了解。
其中一个特别感兴趣的是肠道微生物群与宿主免疫反应在调节肠道炎症方面的相互作用3。肠道微生物群提供一系列免疫刺激分子和抗原，可以激活免疫反应4。虽然效应T细胞和调节性T细胞（Tregs）之间的平衡对于维持肠道稳态至关重要，但肠粘膜CD4 + T细胞对肠道微生物群抗原的过度反应会导致肠道炎症5，6，7。作为一种免疫优势性肠道微生物群抗原，CBir1 鞭毛蛋白与人 CD8，9 的发病机制有关。此外，CBir1 TCR转基因（Tg）T细胞的转移在免疫缺陷小鼠6中诱导肠道炎症，与人类IBD非常相似，表明这种T细胞转移模型有助于研究人类IBD的机制。
这项工作描述了通过通过收养转移CBir1 TCR Tg naϊve CD4 + T细胞并评估疾病严重程度来诱导Rag1-/-小鼠结肠炎的详细方案。此外，还显示了预期的结果，并讨论了程序和故障排除的关键步骤，这将有助于研究人员研究肠道炎症发病机制并测试治疗IBD的潜在药物。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.22 &#181;m vacuum-driven disposable bottle top filter</td>
			<td>MilliporeSigma</td>
			<td>SCGPS05RE</td>
			<td></td>
		</tr>
		<tr>
			<td>100x Penicillin-Streptomycin</td>
			<td>Corning</td>
			<td>30-002-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>100-&#181;m strainer</td>
			<td>BD Biosciences</td>
			<td>352360</td>
			<td></td>
		</tr>
		<tr>
			<td>3-mL Transfer Pipette</td>
			<td>Fisherbrand</td>
			<td>13-711-9CM</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Mouse CD16/32</td>
			<td>Biolegend</td>
			<td>101302</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Mouse CD25-Percp/Cy5.5</td>
			<td>Biolegend</td>
			<td>102030</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse CD3-Percp/Cy5.5</td>
			<td>Biolegend</td>
			<td>100327</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Mouse CD4 APC</td>
			<td>Biolegend</td>
			<td>100516</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Mouse CD4 Magnetic Particles</td>
			<td>BD Biosciences</td>
			<td>551539</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Mouse CD4-BV421</td>
			<td>Biolegend</td>
			<td>100544</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Mouse CD62L-PE</td>
			<td>Biolegend</td>
			<td>104408</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Mouse Foxp3-PE</td>
			<td>ThermoFisher</td>
			<td>12-5773-82</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Mouse IFN&#947;-FITC</td>
			<td>Biolegend</td>
			<td>505806</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Mouse IL-17A-PE/Cy7</td>
			<td>Biolegend</td>
			<td>506922</td>
			<td></td>
		</tr>
		<tr>
			<td>Automated Cell Counter</td>
			<td>Bio-rad</td>
			<td>TC20</td>
			<td></td>
		</tr>
		<tr>
			<td>Brefeldin A</td>
			<td>BD Biosciences</td>
			<td>555029</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Fisher Bioreagents</td>
			<td>BP1600-1</td>
			<td></td>
		</tr>
		<tr>
			<td>C tube</td>
			<td>Miltenyi</td>
			<td>130-093-237</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Separation Magnet</td>
			<td>BD Biosciences</td>
			<td>552311</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase IV</td>
			<td>Sigma-Aldrich</td>
			<td>C5138</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Sigma-Aldrich</td>
			<td>D9542</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissociator Machine</td>
			<td>Miltenyi</td>
			<td>130-096-427</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Corning</td>
			<td>46-034-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA (0.5 M, PH 8.0)</td>
			<td>Corning</td>
			<td>46-034-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>R&#38;D Systems</td>
			<td>S11550</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow cytometer</td>
			<td>BD Biosciences</td>
			<td>LSD Fortessa</td>
			<td></td>
		</tr>
		<tr>
			<td>Heat Lamp</td>
			<td>CoverShield</td>
			<td>BR40</td>
			<td></td>
		</tr>
		<tr>
			<td>Hematoxylin and Eosin (H&#38;E) Stain Kit</td>
			<td>Abcam</td>
			<td>ab245880</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin Syringes</td>
			<td>BD Biosciences</td>
			<td>329412</td>
			<td></td>
		</tr>
		<tr>
			<td>Ionomycin</td>
			<td>ThermoFisher</td>
			<td>I24222</td>
			<td></td>
		</tr>
		<tr>
			<td>Live/dead Fixable Near-IR Dead Cell Stain kit</td>
			<td>ThermoFisher</td>
			<td>L10119</td>
			<td></td>
		</tr>
		<tr>
			<td>MaxQ 6000 Incubated/Refrigerated Stackable Shakers</td>
			<td>ThermoFisher</td>
			<td>SHKE6000</td>
			<td></td>
		</tr>
		<tr>
			<td>NH4Cl</td>
			<td>Thermo Scientific</td>
			<td>A687-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Percoll</td>
			<td>GE Healthcare</td>
			<td>17-0891-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Phorbol-12-myristate 13-acetate</td>
			<td>Sigma-Aldrich</td>
			<td>P8139</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 Medium</td>
			<td>Cytiva HyClone</td>
			<td>SH3002702</td>
			<td></td>
		</tr>
		<tr>
			<td>Sorter</td>
			<td>BD Biosciences</td>
			<td>Arial Fusion</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Automatic Processor</td>
			<td>ThermoFisher</td>
			<td>STP120</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Embedding/Processing Cassette</td>
			<td>Fisher Healthcare</td>
			<td>22048142</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris Base</td>
			<td>Thermo Scientific</td>
			<td>BP154-1</td>
			<td></td>
		</tr>
		<tr>
			<td>True-Nuclear Transcription Factor Buffer Set (including Perm Buffer)</td>
			<td>Biolegend</td>
			<td>424401</td>
			<td></td>
		</tr>
	</tbody>
</table>

induction of intestinal inflammation by adoptive transfer of cbir1 tcr transgenic cd4+ t cells to immunodeficient mice

随着发病率的增加，炎症性肠病（IBD）是影响胃肠道的慢性疾病，给个人和社会带来了相当大的健康和经济负担。因此，研究IBD发病机制和发展的机制至关重要。这里描述了肠道微生物群抗原特异性T细胞转移结肠炎模型。CBir1 鞭毛蛋白已被公认为实验性结肠炎和克罗恩病患者的免疫优势性肠道细菌抗原。CBir1 TCR转基因naϊve CD4 + T细胞，对CBir1鞭毛蛋白具有特异性，可在过继转移到免疫缺陷 的Rag1-/- 小鼠后诱导慢性结肠炎。通过组织病理学评估疾病的严重程度。还测定了固有结肠层中的CD4 + T细胞表型。该模型与IBD的发展非常相似，IBD为研究驱动IBD发病机制和测试治疗IBD的潜在药物提供了理想的小鼠模型。

从成年CBir1 TCR Tg小鼠中分离出每个脾脏约5 x 106个CBir1 TCR Tg naϊve CD4 + T细胞。CBir1 TCR Tg naϊve CD4 + T细胞的转移在受体Rag1-/-小鼠中诱导慢性结肠炎。细胞转移后，监测临床体征以评估肠道炎症的进展，包括体重减轻，粪便稠度和驼背姿势。正如预期的那样，小鼠在细胞转移后三周左右开始减肥，并且在细胞转移后六周体重达到原始重量的80%-85%左右（<strong class=...

Watch this Scientific Journal Video about Induction of Intestinal Inflammation by Adoptive Transfer of CBir1 TCR Transgenic CD4+ T Cells to Immunodeficient Mice at JoVE.com

Induction of Intestinal Inflammation by Adoptive Transfer of CBir1 TCR Transgenic CD4+ T Cells to Immunodeficient Mice

在该协议中，描述了肠道微生物群抗原特异性T细胞过继转移结肠炎模型。从CBir1 TCR转基因小鼠中分离出CD4 + T细胞。这些对于免疫优势性肠道微生物群抗原CBir1鞭毛蛋白具有特异性，其被转移到受体 Rag1-/- 小鼠中，导致肠道炎症。

通过将CBir1 TCR转基因CD4 + T细胞过继转移到免疫缺陷小鼠来诱导肠道炎症

With the increase of incidence, inflammatory bowel diseases (IBD), which are chronic diseases affecting the gastrointestinal tract, impose a considerable ...

炎症性肠病（IBD）给个人和社会带来了相当大的健康和经济负担。该结肠炎模型有助于研究IBD的机制并评估IBD的治疗方法。这种免疫优势性Cbir鞭毛蛋白T细胞特异性过继性转移结肠炎模型提供了对肠道细菌抗原如何诱导T细胞反应以诱导结肠炎的见解。对小鼠实施安乐死后，做一个大约一厘米的左腹部切口，并将皮肤从腹部肌肉组织中拉开。然后，在腹部肌肉组织中做一个大约三厘米的切口。接下来，用无菌剪刀和镊子去除脾脏。将脾脏置于含有五毫升预冷洗涤缓冲液的培养皿中。用两个无菌载玻片的粗糙表面研磨脾脏。通过将细胞悬浮液通过100微米细胞过滤器，将其转移到50毫升离心管中。用五毫升预冷的洗涤缓冲液冲洗载玻片和培养皿，并将洗涤缓冲液转移到管中。离心细胞悬浮液，丢弃上清液，并在每个脾脏中用五毫升预热的Tris氯化铵裂解缓冲液重悬细胞。在室温下孵育10分钟。向管中加入10毫升预冷的洗涤缓冲液。然后，离心细胞悬浮液，弃去上清液，并用10毫升预冷的分离缓冲液重悬细胞。要计数细胞，将细胞悬浮液和台盼蓝充分混合10微升，并将10微升加载到载玻片上。然后，将载玻片插入自动细胞计数器以获得活细胞数。离心剩余的细胞悬浮液并丢弃所有上清液。涡旋抗小鼠CD4磁性颗粒，每1000万个细胞直接加入50微升颗粒，并与细胞沉淀充分混合。将混合物在4摄氏度下孵育30分钟。将细胞颗粒悬浮液转移到无菌收集管中。将3.5毫升预冷的隔离缓冲液加入管中。将管子放在室温下的细胞分离磁铁上八分钟。然后，用三毫升转印移液管小心地吸出上清液。从电池分离磁铁中取出管子。然后，用3.5毫升预冷的分离缓冲液重悬细胞，并将管子在室温下在磁铁上放置四分钟。用3毫升移液管小心地吸出上清液。最后，将细胞重悬于一毫升预冷的FACS缓冲液中。为了将细胞转移到受体小鼠中，将Cbir1 TCR转基因初生CD4阳性T细胞重悬到1x PBS中每毫升500万个细胞。在加热灯下加热小鼠，并使用小鼠约束器约束小鼠。静脉注射200微升细胞悬浮液到小鼠的尾静脉中。对小鼠实施安乐死后，做一个约一厘米的腹侧中线皮肤切口。将皮肤从腹部肌肉组织中拉开。然后，在腹部肌肉组织中做一个三厘米的切口。接下来，识别后部并用无菌剪刀和镊子切除整个结肠。在培养皿中用预冷却的PBS润湿结肠。纵向切开结肠，然后用预冷的PBS冲洗。纵向切除1/3的结肠。将结肠条放在纸巾中，内腔面朝上。使用牙签进行瑞士滚动。将结肠瑞士放入盒中，并将盒放入10%缓冲的福尔马林中24小时。脱水和石蜡包埋结肠与自动处理器。然后，在切片机上切割五微米的组织切片。将组织安装在载玻片上，并进行苏木精和曙红染色。Rag敲除小鼠在细胞转移后三周左右开始减肥，它们的体重在细胞转移后六周达到原始体重的80%至85%左右。受体小鼠在细胞转移后六周显示出较短的结肠长度。受体小鼠在细胞转移后四周在肠道固有层中表现出更多的细胞浸润。他们在细胞转移后五周显示杯状细胞丢失和肠上皮细胞增生，以及细胞转移后六周结肠粘膜下层的粘膜侵蚀和炎性细胞浸润。组织病理学评分从4周大幅增加至6周。同时，单独接受PBS的Rag敲除小鼠没有炎症。使用该程序，结肠细胞因子水平可以通过ELISA和qPCR确定。

Research

JoVE Journal

Medicine

Induction of Intestinal Inflammation by Adoptive Transfer of CBir1 TCR Transgenic CD4+ T Cells to Immunodeficient Mice

Functional Assessment of Intestinal Motility and Gut Wall Inflammation in Rodents: Analyses in a Standardized Model of Intestinal Manipulation

在啮齿类动物的肠动力及胃肠壁的炎症分析功能评价一个标准化的模式肠道操作

Inflammation of the gastrointestinal tract is a common reason for a variety of human diseases. Animal research models are critical in investigating the ...

科研

医学

Neo-Islet Formation in Liver of Diabetic Mice by Helper-dependent Adenoviral Vector-Mediated Gene Transfer

新洲形成的助手依赖型腺病毒载体介导的基因转移对糖尿病小鼠肝

Type 1 diabetes is caused by T cell-mediated autoimmune destruction of insulin-producing cells in the pancreas. Until now insulin replacement is still the ...

Generation of Subcutaneous and Intrahepatic Human Hepatocellular Carcinoma Xenografts in Immunodeficient Mice

皮下及肝内人肝癌裸鼠移植瘤的免疫缺陷小鼠的产生

In  vivo experimental models of hepatocellular carcinoma (HCC) that recapitulate  the human disease provide a valuable platform for research into disease  ...

Induction of Invasive Transitional Cell Bladder Carcinoma in Immune Intact Human MUC1 Transgenic Mice:  A Model for Immunotherapy Development

浸润性膀胱移行细胞癌中的完整的人MUC1转基因小鼠免疫诱导免疫治疗发展模型

A preclinical model of invasive bladder cancer was developed in human mucin 1 (MUC1) transgenic (MUC1.Tg) mice for the purpose of evaluating immunotherapy ...

Murine Endoscopy for In Vivo Multimodal Imaging of Carcinogenesis and Assessment of Intestinal Wound Healing and Inflammation

Murine Endoscopy for In Vivo Multimodal Imaging of Carcinogenesis and Assessment of Intestinal Wound Healing and Inflammation

鼠内镜<em&gt;体内</em&gt;发生和肠道的评价多模式成像伤口愈合和炎症

Mouse models are widely used to study pathogenesis of human diseases and to evaluate diagnostic procedures as well as therapeutic interventions ...

Bile Duct Ligation in Mice: Induction of Inflammatory Liver Injury and Fibrosis by Obstructive Cholestasis

胆管结扎小鼠：感应炎性肝损伤和纤维化的阻塞性胆汁淤积

In most vertebrates, the liver produces bile that is necessary to emulsify absorbed fats and enable the digestion of lipids in the small intestine as well ...

Th17 Inflammation Model of Oropharyngeal Candidiasis in Immunodeficient Mice

口咽念珠菌病的免疫缺陷小鼠Th17细胞炎症模型

Oropharyngeal Candidiasis (OPC) disease is caused not only due to the lack of host immune resistance, but also the absence of appropriate regulation of ...

Dry Powder and Nebulized Aerosol Inhalation of Pharmaceuticals Delivered to Mice Using a Nose-only Exposure System

干粉和药品的雾化吸入气雾剂递送小鼠使用鼻只曝光系统

Obstructive respiratory diseases like asthma and chronic obstructive pulmonary disease (COPD) are currently treated by inhaled anti-inflammatory and ...

Fluorescence-mediated Tomography for the Detection and Quantification of Macrophage-related Murine Intestinal Inflammation

荧光介导的断层扫描对巨噬细胞相关小鼠肠道炎症的检测和定量

Murine models of disease are indispensable to scientific research. However, many diagnostic tools such as endoscopy or tomographic imaging are not ...

Induction of Nephrotic Syndrome in Mice by Retrobulbar Injection of Doxorubicin and Prevention of Volume Retention by Sustained Release Aprotinin

球注射阿霉素对小鼠肾病综合征的诱导作用及缓释抑肽酶预防容积保留率的研究

Nephrotic syndrome is the most extreme manifestation of proteinuric kidney disease and characterized by heavy proteinuria, hypoalbuminemia, and edema due ...