Name: an advanced murine model for nonalcoholic steatohepatitis in association with type 2 diabetes
Uploaded: 2019-04-26T14:30:00
Description: Watch this Scientific Journal Video about An Advanced Murine Model for Nonalcoholic Steatohepatitis in Association with Type 2 Diabetes at JoVE.com

我们感谢安克管辖、戴安娜·沃勒纳、凯瑟琳·威特博士和科内莉亚·赫克曼博士在实验程序方面的协助, 并感谢 Biolegend 的 Benjamin Tiburzy 对门控策略的有益评论。J. s. 得到了赫尔姆霍兹·格兰特 (ICMED) 的支持。这项研究得到了柏林卫生研究所临床研究股的赠款、德国联邦教育和研究部以及爱因斯坦基金会的 "bcrt 赠款" 的支持。K. S.-B。和 h. d. v. 由 FOR2165 提供资金。

这项研究是根据《国家卫生研究院实验动物护理和使用指南》和《动物福利法》在我们的机构动物护理和使用委员会的监督下进行的。动物议定书是根据德国柏林 Charité准则进行的, 并得到 Landesamt für Gesundheit 和 Soziales 的批准, 并遵守了 ARRIVE 准则。
1. 脂肪性肝炎的饮食诱导动物模型
<ol><li>4周时将小鼠 (C57blse-6j, 雄性) 转移到非 spf 住房中, 并将其持续暴露于广泛的环境病原/?...

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脂肪性肝炎与代谢异常 (如肥胖、胰岛素抵抗和血脂异常 15) 有很强的联系。多项研究表明, 脂肪组织炎症会推动2型糖尿病的发病机制, 包括先天免疫系统和适应性免疫系统细胞水平的改变 4,5,16,17.此外, 人们还发现, 肥胖调节免疫途径的激活, 可导致肝脏并发症 18.由?...

肥胖是一种多因素疾病, 也是患心脏病、中风、非酒精性脂肪性肝炎 (nash)、2型糖尿病 (T2D) 和某些类型癌症的主要危险因素。全球肥胖流行率正在迅速上升。如今, 21亿人--占世界人口的近30%--要么肥胖, 要么超重1人。与肥胖相关的胰岛素抵抗可导致 T2D, 当耗尽的胰岛β细胞不能补偿胰岛素维持葡萄糖稳态的需求增加2。
脂肪组织由不同类型的细胞组成, 包括脂肪细胞、内皮细胞、成纤维细胞和免疫细胞。在肥胖的过程中, 免疫细胞数量和活性的变化会导致肥厚性脂肪组织的低度炎症 3,4。具体而言, 人们发现, 过量的能量摄入, 伴随着血糖、甘油三酯和游离脂肪酸水平的长期升高, 会导致脂肪细胞缺氧、内质网应力、线粒体功能受损和增强细胞因子分泌, 导致促炎脂肪免疫细胞激活 5,6。过去的研究主要集中在先天免疫上, 但最近自适应免疫细胞 (T 和 B 细胞) 已成为葡萄糖稳态的重要调节剂。它们具有炎症 (包括 cd8+ t 细胞、th1 和 b 细胞) 或主要的调节功能 (包括调节 t (treg) 细胞、th2 细胞), 既能加剧或抵御胰岛素抵抗7,8,9个。
此外, 还提出了几种机制来解释肥胖如何增加脂肪性肝炎, 包括脂肪组织10产生的细胞因子.NASH 是非酒精性脂肪性肝病的进步形式, 也是发达国家的主要健康负担, 其组织学特征是肝细胞膨胀、脂质积累、纤维化和周细胞炎症, 并可能发展到肝硬化、终末期肝病或肝细胞癌。已知有几种方案 (例如蛋氨酸和胆碱缺乏饮食)可诱导非人类动物模型中的 nash 样肝病理学, 但这些方法大多不能重述 nash 的人类状况及其代谢后果, 因为他们要么需要特定的基因敲除, 非生理饮食操纵或缺乏胰岛素抵抗典型的人类 NASH。此外, 我们对代谢疾病潜在机制的理解目前是基于对在标准特定无病原体 (SPF) 条件下的实验室小鼠进行的实验。这些屏障设施异常卫生, 不考虑人类必须遇到的微生物多样性, 这可能是动物研究翻译到 12、13 临床方法过程中的困难原因,14岁
为了在再生人体免疫条件的高级小鼠模型中, 研究胰岛素抵抗和 NASH 发育过程中脂肪组织和肝脏中不同免疫细胞亚群的情况, 将小鼠置于半无菌的单独笼子中没有障碍的条件。在抗原暴露条件下的小鼠在喂养了13周高脂肪饮食 (hfd) 后, 已经发展了 nash 样的肝脏病理。与年龄匹配的 SPF 小鼠相比, 他们发展了巨囊性脂肪变性, 肝浸润和免疫细胞激活。
这份手稿描述了一个强大的流式细胞仪分析, 以定义和计数免疫细胞亚群从小鼠脂肪组织和肝脏在一个模型的 NASH。流式细胞术分析可以同时检测单个细胞的多个参数, 而不是 RT-PCR 或免疫组织化学方法。
总之, 我们的研究提供了一个小鼠模型的短期 HFD 调查胰岛素抵抗和 NASH 的发展, 以及潜在的机制, 也显示了对人类状况的忠诚度。

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			<td>60 kJ% from fat, 19&#8201;kJ% from proteins, and 21&#8201;kJ% from carbohydrates</td>
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			<td style="width:119px;">S3146</td>
			<td>used for dissection purposes&#160;</td>
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			<td height="21" style="height:21px;width:351px;">PE anti-mouse CD25 Antibody</td>
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			<td>AB_312846 (BioLegend Cat. No. 101903)</td>
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			<td style="width:169px;">Biolegend&#160;</td>
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			<td>AB_313102 (BioLegend Cat. No. 104417)</td>
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			<td height="21" style="height:21px;width:351px;">PE/Cy7 anti-mouse I-A/I-E (MHCII) Antibody</td>
			<td style="width:169px;">Biolegend&#160;</td>
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			<td>AB_2290801 (BioLegend Cat. No. 107629)</td>
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			<td height="21" style="height:21px;width:351px;">PE/Dazzle 594 anti-mouse CD4 Antibody</td>
			<td style="width:169px;">Biolegend&#160;</td>
			<td style="width:119px;">100565</td>
			<td>AB_2563684 (BioLegend Cat. No. 100565)</td>
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			<td height="21" style="height:21px;width:351px;">PerCP/Cy5.5 anti-mouse CD44 Antibody</td>
			<td style="width:169px;">Biolegend&#160;</td>
			<td style="width:119px;">103031</td>
			<td>AB_2076206 (BioLegend Cat. No. 103031)</td>
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			<td height="21" style="height:21px;width:351px;">PerCP/Cy5.5 anti-mouse Gr-1 Antibody</td>
			<td style="width:169px;">Biolegend&#160;</td>
			<td style="width:119px;">108427</td>
			<td>AB_893561 (BioLegend Cat. No. 108427)</td>
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			<td height="21" style="height:21px;width:351px;">Phosphate buffered saline&#160;</td>
			<td style="width:169px;">Gibco</td>
			<td style="width:119px;">12559069</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Round-Bottom Tubes with cell strainer cap</td>
			<td style="width:169px;">STEMCELL Technologies&#160;</td>
			<td style="width:119px;">38030</td>
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			<td height="21" style="height:21px;width:351px;">TruStain fcX anti-mouse CD16/32</td>
			<td style="width:169px;">Biolegend&#160;</td>
			<td style="width:119px;">101301</td>
			<td>AB_312800 (BioLegend Cat. No. 101301)</td>
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			<td height="21" style="height:21px;width:351px;">Trypan Blue&#160;</td>
			<td style="width:169px;">Sigma-Aldrich&#160;</td>
			<td style="width:119px;">T6146</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Zombie NIR Fixable Viability Kit</td>
			<td style="width:169px;">Biolegend&#160;</td>
			<td style="width:119px;">423105</td>
			<td>viablity stain&#160;</td>
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an advanced murine model for nonalcoholic steatohepatitis in association with type 2 diabetes

肥胖与慢性低度炎症和胰岛素抵抗相关, 导致慢性代谢疾病 (如2型糖尿病和非酒精性脂肪性肝炎 (NASH) 发病率不断上升。最近的研究已经证实, 促炎性免疫细胞浸润肥胖肥厚脂肪组织和肝脏。鉴于免疫细胞在代谢稳态方面的重要性正在显现, 在2型糖尿病和 NASH 的发展过程中, 迫切需要量化和描述免疫细胞的修饰。然而, 诱导人类纳什典型的病理生理特征的动物模型却很少。
在本文中, 我们提供了一个详细的协议, 以识别从肝脏和脂肪组织分离出的免疫细胞亚群在一个可靠的小鼠模型的 NASH, 建立通过住房高脂肪饮食 (HFD) 小鼠在非特定病原 (SPF) 条件下没有障碍至少七个星期。我们演示了小鼠在非 spf 条件下的处理, 组织的消化和巨噬细胞的鉴定, 自然杀伤细胞, 树突状细胞, B 和 T 细胞子群流式细胞仪。提供了来自 SPF HFD 小鼠和非 spf 小鼠的代表性流式细胞仪图。为了获得可靠和可解释的数据, 使用抗体、准确和精确的组织消化方法以及流式细胞术中的适当门控是关键要素。
通过将小鼠安置在非 spf 条件下和不特异性接触微生物抗原来恢复其生理抗原暴露的干预措施, 可以为调查饮食诱导的免疫改变之间的联系提供相关的工具肥胖和相关的长期并发症。

所描述的协议允许在饮食诱导的 NASH 模型中表征从小鼠外侧脂肪组织和肝脏中分离出的先天和适应性免疫细胞的表面标记。在这种模型中, NASH 是由 HFD 加蔗糖的给药诱导的 (6%)在 c57bl过来6j 小鼠的饮用水中摄入 7至15周, 如先前报道的 13周。重要的是, 小鼠被安置在半无菌条件下, 因此在整个实验过程中接触环境抗原。HFD 喂养的是生活在 SPF 条件下的老鼠, 而在 SPF 和非 spf 条件下的?...

Watch this Scientific Journal Video about An Advanced Murine Model for Nonalcoholic Steatohepatitis in Association with Type 2 Diabetes at JoVE.com

An Advanced Murine Model for Nonalcoholic Steatohepatitis in Association with Type 2 Diabetes

描述了一种简单可靠的饮食诱导的非酒精性脂肪性肝炎 (NASH) 啮齿类动物模型, 通过动物的非 spf 外壳和特定高脂肪饮食的管理实现。我们描述了肝脏和脂肪免疫细胞亚群的鉴定, 通过使小鼠接触环境细菌来重述人类的免疫状况。

非酒精性脂肪性肝炎合并2型糖尿病的一种先进的小鼠模型

Obesity is associated with chronic low-grade inflammation and insulin resistance, contributing to an increasing prevalence of chronic metabolic diseases, ...

这种方法可以帮助回答免疫代谢领域关于免疫细胞对脂肪和肝组织中脂肪性腺炎的肥胖引起的炎症的影响的问题。这些技术已经优化，在通过非特异性无病原体的啮齿动物外壳获得的非酒精性脂肪性脂肪性肝炎饮食诱导模型中分离脂肪和肝免疫细胞。这些方法可以帮助加深我们对维持代谢平衡的免疫机制的理解。一般来说，这种技术的新个体将难以最大限度地提高功能上可行的分离细胞的产量，并在流式细胞学中建立适当的浇注。为了产生脂肪组织单细胞悬浮液，用70%乙醇喷洒安乐死小鼠的胸部，并小心地通过肋骨笼的整个长度的内膜和腹壁进行五到六厘米的中央切口，以暴露胸腔和心脏。使用 26 量针，将至少 10 毫升 0.9% 盐水溶液注射到左心室的顶点，然后用剪刀打开腹腔。然后，解剖表观性脂肪组织，并称量它。在将组织片段转移到50毫升的圆锥管中之前，在4摄氏度的培养皿中机械地将脂肪组织分离成细片。在PBS中用0.5%牛血清白蛋白或BSA的1毫升冲洗培养皿，并在管中加入三毫升新鲜准备的脂肪组织消化液。在37摄氏度和每分钟200次旋转下20分钟后，在PBS中加入5毫升0.5%BSA，并将脂肪消化池放在冰上。使用 10 毫升血清移液器多次对溶液进行三次三化，并使用柱塞通过 100 微米应变器将溶液传递。通过离心分离组织分数，并使用移液器丢弃浮动的脂肪细胞分数。将血管小分细胞颗粒重新吸收在一毫升氯化铵-钾或CK，解液缓冲液中，几秒钟后用10毫升2%胎儿小牛血清或FCS停止解解反应。然后，通过离心收集分离的白血球，并在PBS中250微升2%FCS中重新暂停颗粒进行计数。在使用注射器盖章在四摄氏度的培养皿中机械分离组织之前，将肝脏收获成冰上PBS的锥形管。将解剖的肝脏组织片段转移到含有10毫升热肝消化液的50毫升管中，然后用3毫升新鲜肝脏消化液冲洗培养皿。在37摄氏度和每分钟200次旋转下20分钟后，用20毫升的HSS停止消化，用10毫升血清移液器多次三重奏组织浆。使用柱塞通过100微米过滤器传递组织溶液，并通过离心收集肝细胞。将颗粒重新在20毫升新鲜HSS中，通过离心去除肝细胞基质。收集离心的上一液，在HSSS中33%的低粘度密度梯度介质溶液的10毫升中重新填充颗粒。通过密度梯度离心分离肝脏免疫细胞，将白细胞颗粒重新在一毫升的CK解菌缓冲液中。在室温下4分钟后，用10毫升HSS停止解解，并通过30微米孔隙过滤器将细胞过滤到15毫升管中进行离心。然后，在 PBS 中 250 微升 2%FCS 中重新暂停颗粒进行计数。对于分离的白细胞群的流动细胞学分析，将PBS中2%FCS的100微升中6个细胞的3倍10，从感兴趣的组织到单个聚苯乙烯荧光活性细胞分选，或FACS，管。在每个管子上加入10微升的抗CD16/CD32抗体，在冰上阻断任何非特异性结合10分钟，然后用适当体积的抗体混合物染色，每个样品1微升的活体染料，允许活细胞和死细胞分立。在4摄氏度下20分钟后，用两毫升2%FCS加PBS每次洗涤两次，以300倍g的离心机在4摄氏度下洗涤5分钟。然后，在新鲜的 PBS 加 FCS 中重新暂停颗粒。要通过流式细胞分析细胞，请使用未污染的负对量样本设置正向和侧散射，并调整流式细胞仪的电压，以检测可行的白细胞群体并排除碎屑。接下来，在运行实验样品之前，运行单色控制样本进行多色补偿，收集每管至少五乘十到四个事件。然后，导出FCS数据文件进行分析，并设置CD45阳性白细胞的浇注策略，以便识别随后感兴趣的免疫细胞群。在这里，显示了T细胞亚细胞的代表性的浇注策略，包括双核歧视和在穆林性脂肪中的生存能力染色。CD45阳性白细胞首先被封闭为CD4和CD8，随后为CD44和CD62配体，以区分天真、中央记忆、效应器记忆和效应器T细胞。然后CD44阳性细胞进一步特征为CD127，杀手细胞卵素样受体G1，和编程死亡-1。在这里，显示了分析B细胞、粒细胞、自然杀伤细胞、巨噬细胞和树突状细胞的浇注策略。在非SPF条件下的高脂肪饮食小鼠中，可检测到效应者记忆CD4阳性和CD8阳性细胞的较高百分比，而与第7周的SPF高脂肪饮食小鼠相比，这些动物的肝内天真CD4阳性和CD8阳性T细胞的比例要低得多。严重的性肺病，包括大脂液滴的增加，导致大脂球菌，球状炎症，肝细胞膨胀，和破坏球状结构发现在高脂肪饮食暴露的小鼠，而只有一些SPF小鼠显示轻微的脂肪积累在肝脏。此外，在高脂肪饮食暴露小鼠的食肉脂肪组织中，自然杀伤细胞的比例较高，而高脂肪饮食SPF小鼠的树突状细胞比例较高。尝试此过程时，除非另有说明，否则保持细胞在冰上和黑暗中非常重要。现在，您应该对如何正确准备单细胞悬浮液以及如何分离粘液和肝组织进行流动细胞学实验有了很好的理解。

Research

JoVE Journal

Immunology and Infection

Trichuris muris Infection: A Model of Type 2 Immunity and Inflammation in the Gut

Trichuris muris Infection: A Model of Type 2 Immunity and Inflammation in the Gut

鞭虫感染muris：在肠道内的2型免疫和炎症模型

Trichuris muris is a natural pathogen of mice and is biologically and antigenically similar to species of Trichuris that 
infect humans and livestock1. ...

科研

免疫与感染

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

主要鼠Ⅱ型肺泡上皮细胞的功能及分子生物学研究的流式细胞仪分离

Throughout  the last years, the contribution of alveolar type II epithelial cells (AECII)  to various aspects of immune regulation in the lung has been ...

Accelerated Type 1 Diabetes Induction in Mice by Adoptive Transfer of Diabetogenic CD4+ T Cells

加速1型糖尿病感应在小鼠体内的过继转移致糖尿病的CD4 + T细胞

The nonobese diabetic (NOD) mouse spontaneously develops autoimmune diabetes after 12 weeks of age and is the most extensively studied animal model of ...

Use of Image Cytometry for Quantification of Pathogenic Fungi in Association with Host Cells

使用的细胞图像分析仪量化致病真菌与宿主细胞协会

Studies of the cellular pathogenesis mechanisms of pathogenic yeasts such as Candida albicans, Histoplasma capsulatum, and Cryptococcus neoformans ...

Fluorescence Microscopy Methods for Determining the Viability of Bacteria in Association with Mammalian Cells

荧光显微镜方法测定细菌在协会的可行性与哺乳动物细胞

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. Current protocols ...

Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

隔离和小鼠骨髓的静脉注射单核细胞衍生

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of ...

Ex Vivo Infection of Murine Epidermis with Herpes Simplex Virus Type 1

Ex Vivo Infection of Murine Epidermis with Herpes Simplex Virus Type 1

鼠表皮离体感染单纯疱疹病毒1型

To enter its human host, herpes simplex virus type 1 (HSV-1) must overcome the barrier of mucosal surfaces, skin, or cornea. HSV-1 targets keratinocytes ...

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

使用的淋巴细胞外渗分析<em&gt;体外</em&gt;人类血脑屏障模型

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte ...

Application of Consistent Massage-Like Perturbations on Mouse Calves and Monitoring the Resulting Intramuscular Pressure Changes

在小鼠小腿上应用一致的按摩样扰动，并监测由此产生的肌肉压力变化

Massage is generally recognized to be beneficial for relieving pain and inflammation. Although previous studies have reported anti-inflammatory effects of ...

High-Efficiency Generation of Antigen-Specific Primary Mouse Cytotoxic T Cells for Functional Testing in an Autoimmune Diabetes Model

在自身免疫糖尿病模型中高效生成抗原特异性原小鼠细胞毒性T细胞细胞细胞，用于功能测试

Type 1 Diabetes (T1D) is characterized by islet-specific autoimmunity leading to beta cell destruction and absolute loss of insulin production. In the ...