Name: assessment of antibody-based drugs effects on murine bone marrow and peritoneal macrophage activation
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力是不列颠哥伦比亚大学4年奖学金 (4YF) 研究生李兆基奖获得者。L.M.S. 是加拿大胃肠病学/克罗恩病和结肠炎加拿大协会/研究院新调查员薪资奖的接受者, 是迈克尔. 史密斯健康研究基金会的生物医学学者。这项工作得到加拿大血液服务项目赠款的支持, 与加拿大卫生研究所合作 (赠款 CIHR2016-LS)。

这里描述的所有方法都得到了不列颠哥伦比亚大学机构动物保育和使用委员会 (IACUC) 的批准。
1. 骨髓巨噬细胞的抗体衍生与活化
<ol>
	<li>使用 CO2窒息执行安乐死。<ol>
		<li>将鼠标置于感应腔内。安乐小鼠与5% 异氟醚麻醉, 60-九十年代, 直到静止和呼吸是深和缓慢。</li>
		<li>关闭异氟醚麻醉并管理 CO2的 6-8 升/分, 直到鼠标停止呼吸。将鼠标放在 CO2上, 至...

<ol>
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巨噬细胞活化状态在组织稳态和疾病中起重要作用22。巨噬细胞可以有不同的和重叠的活化状态, 根据他们的微环境的提示3。它们在炎症反应的各个阶段都有不同的作用: 防御病原体, 伤口愈合和组织恢复, 也有明显的抗炎活化状态, 对于关闭炎症反应很重要2.抗炎巨噬细胞激活需要两个外部刺激巨噬细胞产生大量的抗炎细胞因子, IL-10, 和低量的?...

巨噬细胞是先天免疫的, 在免疫应答中扮演多重角色, 感染或受伤。巨噬细胞负责启动免疫应答感染或组织损伤, 停止炎症反应, 并促进愈合响应1。三最佳研究的巨噬细胞活化状态的例子是: 1) 巨噬细胞治疗干扰素伽玛 (IFNγ) 和细菌脂多糖 (lps), 指定 M (IFNγ + lps), 导致炎症反应;2) 用白细胞介素 4 (IL-4)、M (IL-4) 刺激的巨噬细胞, 与愈合应答有关;3) 巨噬细胞刺激与免疫复合体 (ic) 和 lps, M (ic + lps), 有能力关闭炎症反应2,3。m (IC + LPS) 是不同的 m (IL-4) 伤口愈合巨噬细胞, 并没有表达的酶酸 (Arg-1) 或 FIZZ14。这些抗炎巨噬细胞的最佳标记是其细胞因子的生成5。巨噬细胞在维持健康方面有多种作用, 但也会导致炎症性疾病和癌症3。因此, 巨噬细胞是治疗各种疾病的关键治疗靶。重要的是要研究抗体对其活化状态的影响, 发展 macrophage-based 治疗疾病。
本文的重点是利用小鼠骨髓源性巨噬细胞 (BMDMs) 和腹腔巨噬细胞检测抗体药物对炎症反应的影响体外和体内。最近, 有多项研究显示抗体对巨噬细胞激活的影响6,7,8。巨噬细胞 co-activated 与免疫配合物, 是抗体与抗原复合, 和 LPS, 通常炎症刺激, 产生非常高水平的抗炎细胞因子, IL-10, 和非常低的水平的炎细胞因子,白细胞介素 12 (IL-12)9。此外, 昔, 一个抗 TNFα单克隆抗体, 已发现工作, 部分, 通过诱导抗炎巨噬细胞通过其片段结晶 (Fc) 区域7。我们已经报告, 丙种球蛋白 + lps 诱导抗炎巨噬细胞活化, 类似于 M (IC + lps), 其中 co-stimulated 巨噬细胞产生大量的 IL-10 和低量的炎细胞因子亚基白细胞介素12或 23 p40 (IL-12/23p40), 白细胞介素-6 (IL-6), 和 TNF8。丙种球蛋白是一种由多克隆抗体组成的药物, 主要是 IgG, 从1000多个捐献者的血液中汇集而来10。它被用于治疗各种各样的免疫疾病, 例如特发性血小板减少紫癜和慢性脱髓鞘神经病, 但它的作用机制不完全地被了解11。抗体基础药物对巨噬细胞活化的影响可以用本文所描述的方法进行评估。
特定生物制剂对巨噬细胞活化的影响可在 BMDMs 和腹腔巨噬细胞中检测。使用这些巨噬细胞来源, 可对原体细胞进行评估。对原代细胞抗体的初步检测比其他耗时和昂贵的疾病模型需要更少的时间和金钱投资。通过将药物注入健康的鼠标在体内, 并隔离这些细胞并分析它们的体外, 可以确定是否有必要进行研究以评估生物制剂是否会影响巨噬细胞在疾病模型中的活化作用. 
很少有研究测试生物疗法对巨噬细胞活化的影响在体外和在体内直接, 我们的技术提供了比替代技术的优势。目前的技术包括测试生物药物对混合细胞数量的影响在体外, 如昔在混合淋巴细胞反应 (MLR) 或静脉注射丙种球蛋白对人巨噬细胞外周血单个核的影响不能归因于特定的单元格类型7,12。使用 BMDMs 和腹腔巨噬细胞是有利的, 在使用单元格线, 如 RAW264.7 细胞, 不产生炎细胞因子, IL-12, 以响应 LPS8,13。检测抗体药物对巨噬细胞反应的影响ex 体内具有优势, 因为细胞因子反应可以直接归因于巨噬细胞, 而不是通过测量血清细胞因子水平来推断巨噬细胞的反应,14.BMDMs 和腹腔巨噬细胞可以获得和分离的基因修饰小鼠, 以确定特定的作用, 一个蛋白质的抗炎巨噬细胞活化。例如, 我们使用了Il10缺陷的(--) BMDMs 来证明静脉注射丙种球蛋白诱导的炎细胞因子的减少是部分依赖于 IL-108。药物的作用机制可以用西方印迹来进行研究, 在那里可以确定特定蛋白质和信号事件的角色。定量聚合酶链反应 (qPCR) 可以在 BMDMs 或腹腔巨噬细胞上进行, 以显示抗体活化引起的基因表达模式。小鼠疾病模型可以提供关于抗体生物疗法在炎症性肠病、类风湿关节炎和癌症等疾病模型中的潜在功效的信息15,16,17. 然而, 这里描述的技术将提供关于这些生物制剂的作用机制的信息通过确定他们是否导致抗炎巨噬细胞活动。

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Iscove&#8217;s modified Dulbecco&#8217;s medium (IMDM)</td>
			<td style="width:209px;">Life technologies</td>
			<td style="width:408px;">12440053</td>
			<td style="width:241px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Fetal Bovine Serum (FBS)</td>
			<td style="width:209px;">Life technologies</td>
			<td style="width:408px;">12483-020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Recombinant murine macrophage colony stimulating factor (MCSF)</td>
			<td style="width:209px;">Stemcell technologies</td>
			<td>78059</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Penicillin-streptomycin</td>
			<td style="width:209px;">Life technologies</td>
			<td style="width:408px;">15140148</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Monothioglycerol (MTG)</td>
			<td style="width:209px;">Sigma</td>
			<td>88640</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">1X red blood cell lysis buffer</td>
			<td style="width:209px;">eBioscience</td>
			<td style="width:408px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Cell dissociation buffer</td>
			<td style="width:209px;">Life technologies</td>
			<td style="width:408px;">13150016</td>
			<td>Enzyme-Free, Hanks&#39;s-based, EDTA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Lipopolysaccharide (LPS)</td>
			<td style="width:209px;">Sigma aldrich</td>
			<td style="width:408px;">L 4516</td>
			<td>From E. coli 0127:B8</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">IVIg (Gammunex)</td>
			<td style="width:209px;">Grifols</td>
			<td>Received from BC Children&#39;s Hospital, Transfusion Medicine</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">IVIg (Gammagard liquid)</td>
			<td style="width:209px;">Baxter Healthcare Corporation</td>
			<td>Received from BC Children&#39;s Hospital, Transfusion Medicine</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">IVIg (Octagam)</td>
			<td style="width:209px;">Octapharma</td>
			<td>Received from BC Children&#39;s Hospital, Transfusion Medicine</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Phosphate buffered saline (PBS) (sterile), pH 7.4</td>
			<td style="width:209px;">Life technologies</td>
			<td style="width:408px;">10010023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">mouse IL-10 ELISA</td>
			<td style="width:209px;">BD biosciences</td>
			<td style="width:408px;">555252</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">mouse IL-12/23p40 ELISA</td>
			<td style="width:209px;">BD biosciences</td>
			<td style="width:408px;">555165</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">anti-Erk1/2 antibody</td>
			<td style="width:209px;">Cell signalling technology</td>
			<td style="width:408px;">9101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">anti-pp38 antibody</td>
			<td style="width:209px;">Cell signalling technology</td>
			<td style="width:408px;">9211</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">anti-GAPDH antibody</td>
			<td style="width:209px;">Fitzgerald industries</td>
			<td style="width:408px;">10R-G109A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">26 g needle</td>
			<td style="width:209px;">BD biosciences</td>
			<td style="width:408px;">305110</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">1 mL syringe</td>
			<td style="width:209px;">BD biosciences</td>
			<td style="width:408px;">309659</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">10 mL syringe</td>
			<td style="width:209px;">BD biosciences</td>
			<td style="width:408px;">309604</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">15 mL conical tube</td>
			<td style="width:209px;">BD biosciences</td>
			<td style="width:408px;">352096</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">50 mL conical tube</td>
			<td style="width:209px;">BD biosciences</td>
			<td style="width:408px;">352070</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">microcentrifuge tube (1.7 mL)</td>
			<td style="width:209px;">Diamed</td>
			<td style="width:408px;">SPE155-N</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:453px;">75 cm2 tissue culture treated flask</td>
			<td style="width:209px;">BD biosciences</td>
			<td style="width:408px;">353136</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Cell scraper</td>
			<td style="width:209px;">BD biosciences</td>
			<td style="width:408px;">353085</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Forcepts</td>
			<td style="width:209px;">VWR</td>
			<td>82027-386</td>
			<td>fine tip, dissecting&#160;</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:453px;">Scissors</td>
			<td style="width:209px;">VWR</td>
			<td>82027-582</td>
			<td>Delicate, 4 1/2&#34;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Brightfield microscope</td>
			<td style="width:209px;">Motic</td>
			<td style="width:408px;">AE31</td>
			<td>Inverted phase contrast</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Scale&#160;</td>
			<td style="width:209px;">Mettler&#160;</td>
			<td style="width:408px;">PE 3000</td>
			<td></td>
		</tr>
	</tbody>
</table>

assessment of antibody-based drugs effects on murine bone marrow and peritoneal macrophage activation

巨噬细胞是吞噬先天免疫细胞, 它启动免疫反应的病原体和贡献愈合和组织恢复。巨噬细胞在关闭炎症反应方面同样重要。我们已经表明, 静脉注射免疫球蛋白 (丙种球蛋白) 刺激的巨噬细胞可以产生大量的抗炎细胞因子, 白细胞介素 10 (IL-10), 和低水平的炎细胞因子响应细菌多糖 (LPS)。丙种球蛋白是一种多抗体, 主要是免疫球蛋白 Gs (IgGs), 从血浆中汇集超过1000献血者。它是用来补充免疫缺陷患者的抗体或抑制免疫反应的患者自身免疫或炎症条件。昔, 一个治疗性抗肿瘤坏死因子α (TNFα) 抗体, 也被证明激活巨噬细胞产生 IL-10 反应炎症刺激。免疫球蛋白和其他抗体生物制剂可以通过测试来确定它们对巨噬细胞活化的影响。本文介绍了在体外抗体激活的小鼠骨髓巨噬细胞的衍生、刺激和评价方法, 以及抗体激活的小鼠腹腔巨噬细胞 (体内。最后, 我们展示了利用西方印迹来确定特定细胞信号通路对抗炎巨噬细胞活动的贡献。这些协议可以与基因修饰的小鼠一起使用, 以确定特定的蛋白质对抗炎巨噬细胞活化的影响。这些技术也可以用来评估特定的生物制品是否可以通过改变巨噬细胞的 IL-10-producing 抗炎活化状态, 减少炎症反应在体内。这可以提供关于巨噬细胞活化在动物疾病模型中的作用的信息, 并提供对人类潜在的新的行动机制的洞察力。反之, 这可能告诫不要使用特定的抗体生物制剂来治疗传染性疾病, 特别是如果巨噬细胞在宿主防御感染中起重要作用。

小鼠骨髓源性巨噬细胞可以从骨髓物的造血细胞前体中培养。骨髓物池从股骨和胫骨的一个 C57BL/6 鼠通常产生 107骨髓衍生巨噬细胞, 使他们成为一个方便的来源的巨噬细胞的实验。BMDMs 可用于测试抗体的药物反应时, 挑战与炎症刺激体外。图 1显示了丙种球蛋白品牌, Gammunex (广西), + lps 增加的抗炎细胞因子的生产, IL-10, 7 倍与 lps 刺激单...

Watch this Scientific Journal Video about Assessment of Antibody-based Drugs Effects on Murine Bone Marrow and Peritoneal Macrophage Activation at JoVE.com

Assessment of Antibody-based Drugs Effects on Murine Bone Marrow and Peritoneal Macrophage Activation

抗体药物对炎症性疾病进行了革命性的治疗。除了对特定靶点产生直接影响外, 抗体还能激活巨噬细胞成为抗炎药。该协议描述了如何抗炎巨噬细胞活化可以评估在体外,使用小鼠骨髓巨噬细胞, 和在体内,使用腹腔巨噬细胞。

抗体药物对小鼠骨髓及腹腔巨噬细胞活化作用的评价

Macrophages are phagocytic innate immune cells, which initiate immune responses to pathogens and contribute to healing and tissue restitution. Macrophages ...

Research

JoVE Journal

Immunology and Infection

Murine Model of CD40-activation of B cells

激活的B细胞CD40的小鼠模型

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand ...

科研

免疫与感染

Generation and Labeling of Murine Bone Marrow-derived Dendritic Cells with Qdot Nanocrystals for Tracking Studies

小鼠骨髓来源的树突状细胞与跟踪研究的Qdot纳米晶的生成及标签

Dendritic cells (DCs) are professional antigen presenting cells (APCs) found in peripheral tissues and in immunological organs such as thymus, bone ...

Investigation of Macrophage Polarization Using Bone Marrow Derived Macrophages

使用骨髓源性巨噬细胞巨噬细胞极化的调查

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic ...

Bone Marrow-derived Macrophage Production

骨髓衍生的巨噬细胞产

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because ...

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 ...

Tracking Mouse Bone Marrow Monocytes In Vivo

Tracking Mouse Bone Marrow Monocytes In Vivo

跟踪小鼠骨髓单核细胞<em&gt;在体内</em

Real time multiphoton imaging provides a great opportunity to study cell trafficking and cell-to-cell interactions in their physiological 3-dimensionnal ...

Murine Hind Limb Long Bone Dissection and Bone Marrow Isolation

鼠后肢长骨解剖和骨髓隔离

Investigation of the bone and the bone marrow is critical in many research fields including basic bone biology, immunology, hematology, cancer metastasis, ...

Femur Window Chamber Model for In Vivo Cell Tracking in the Murine Bone Marrow

Femur Window Chamber Model for In Vivo Cell Tracking in the Murine Bone Marrow

股骨窗口商会模型<em&gt;在体内</em在小鼠骨髓&gt;手机跟踪

Bone marrow is a complex organ that contains various hematopoietic and non-hematopoietic cells. These cells are involved in many biological processes, ...

Isolation and Activation of Murine Lymphocytes

分离及小鼠淋巴细胞的激活

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading ...

Flow Cytometric Analysis of Particle-bound Bet v 1 Allergen in PM10

粒子结合Bet v 1的变应原PM10的流式细胞分析

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in ...