Name: adoptive transfer of il-33-stimulated macrophages into bleomycin-induced mouse models to study their effect on idiopathic pulmonary fibrosis in vivo
Uploaded: 2023-05-05T13:30:00
Description: Watch this Scientific Journal Video about Adoptive Transfer of IL-33-Stimulated Macrophages into Bleomycin-Induced Mouse Models to Study Their Effect on Idiopathic Pulmonary Fibrosis In Vivo at JoVE.c

作者认可江南大学实验室管理专题：基于病理标本的数字切片库构建（JDSYS202223）和国家自然科学基金（81800065）。

所有实验均按照《实验动物护理和使用指南》进行。所有动物实验均经江南大学实验动物福利伦理委员会批准（JN No. 20211130m1720615[501]）。
注意：本研究总共使用了10只年龄在6-8周大，体重20-25g的雄性C57BL / 6小鼠。研究中的三个实验组各包括三只受体小鼠，一只宿主小鼠用于IM分离。
1.小鼠肺巨噬细胞耗竭
<ol><li>提前30分钟从冰箱中取...

<ol>
	<li>Heukels, P., Moor, C. C., vonder Th&#252;sen, J. H., Wijsenbeek, M. S., Kool, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammation+and+immunity+in+IPF+pathogenesis+and+treatment.">Inflammation and immunity in IPF pathogenesis and treatment.</a> Respiratory Medicine. 147, 79-91 (2019).</li><li>Zhang, Y., Zhang, Y., Li, X., Zhang, M., Lv, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microarray+analysis+of+circular+RNA+expression+patterns+in+polarized+macrophages.">Microarray analysis of circular RNA expression patterns in polarized macrophages.</a> International Journal of Molecular Medicine. 39 (2), 373-379 (2017).</li><li>Lee, J. 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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=Interleukin-33+potentiates+bleomycin-induced+lung+injury.">Interleukin-33 potentiates bleomycin-induced lung injury.</a> American Journal of Respiratory Cell and Molecular Biology. 49 (6), 999-1008 (2013).</li><li>Nie, Y., 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=AKT2+regulates+pulmonary+inflammation+and+fibrosis+via+modulating+macrophage+activation.">AKT2 regulates pulmonary inflammation and fibrosis via modulating macrophage activation.</a> Journal of Immunology. 198 (11), 4470-4480 (2017).</li><li>Qian, 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=The+transcription+factor+PU.1+promotes+alternative+macrophage+polarization+and+asthmatic+airway+inflammation.">The transcription factor PU.1 promotes alternative macrophage polarization and asthmatic airway inflammation.</a> Journal of Molecular Cell Biology. 7 (6), 557-567 (2015).</li><li>Shah, S., Dhawan, V., Holm, R., Nagarsenker, M. 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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulmonary+macrophages:+A+new+therapeutic+pathway+in+fibrosing+lung+disease.">Pulmonary macrophages: A new therapeutic pathway in fibrosing lung disease.</a> Trends in Molecular Medicine. 22 (4), 303-316 (2016).</li><li>Wendisch, D., 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=SARS-CoV-2+infection+triggers+profibrotic+macrophage+responses+and+lung+fibrosis.">SARS-CoV-2 infection triggers profibrotic macrophage responses and lung fibrosis.</a> Cell. 184 (26), 6243-6261 (2021).</li><li>Zhang, 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=Macrophages:+Friend+or+foe+in+idiopathic+pulmonary+fibrosis.">Macrophages: Friend or foe in idiopathic pulmonary fibrosis.</a> Respiratory Research. 19 (1), 170 (2018).</li></ol>

本研究为去除、分离、培养和转移巨噬细胞提供了一种有效的方法，有助于研究小鼠肺纤维化的机制。小鼠巨噬细胞耗竭的方法很多，如气管给药、尾静脉注射和鼻吸11。本研究优化了鼻吸法，操作简单，可有效耗竭肺巨噬细胞8，9。在IL-33刺激培养中的IMs24小时后，使用非侵入性气管给药途径将IM过继转移到受体小鼠的肺部，这对小鼠?...

An erratum was issued for:&#160;<a href="https://www.jove.com/t/64742">Adoptive Transfer of IL-33-Stimulated Macrophages into Bleomycin-Induced Mouse Models to Study Their Effect on Idiopathic Pulmonary Fibrosis In Vivo</a>. The Protocol section was updated.
Step 2.1 of the Protocol was updated from:
Anesthetize the host mouse with 3% isoflurane, and then euthanize it by cervical dislocation. Disinfect the skin of the euthanized mouse with 75% alcohol and iodine. Use scissors to cut through the skin and expose the cardiopulmonary tissue
to:
Anesthetize the host mouse&#160;with an intraperitoneal injection of Ketamine (120 mg/kg) and Xylazine (16 mg/kg). Confirm the depth of anesthesia via loss of the toe pinch reflex. Apply veterinary ointment to both eyes to prevent dryness under anesthesia. Then, disinfect the skin of the anesthetized mouse with 75% alcohol and iodine. Use scissors to cut through the skin and expose the cardiopulmonary tissue

An erratum was issued for:&#160;<a href="https://www.jove.com/t/64742">Adoptive Transfer of IL-33-Stimulated Macrophages into Bleomycin-Induced Mouse Models to Study Their Effect on Idiopathic Pulmonary Fibrosis In Vivo</a>. The Protocol section was updated.

Erratum: Adoptive Transfer of IL-33-Stimulated Macrophages into Bleomycin-Induced Mouse Models to Study Their Effect on Idiopathic Pulmonary Fibrosis In Vivo

特发性肺纤维化（IPF）是一种由多种因素引起的弥漫性肺部炎症性疾病1。在Th1和Th2免疫反应的细胞因子微环境中，巨噬细胞可以极化为经典活化的巨噬细胞（M1）和替代活化的巨噬细胞（M2）。脂多糖（LPS）或细胞因子IFN-γ诱导M1巨噬细胞极化并产生促炎细胞因子，包括iNOS，IL-1，IL-6，TNF-α和IL-12。相比之下，II型细胞因子IL-4和IL-13驱动M2巨噬细胞的极化，M2巨噬细胞可以产生不同的成纤维细胞生长促进因子，如TGF-β和PDGF，促进肺纤维化2。IPF的病理过程伴随着巨噬细胞活化和浸润。IPF通过释放细胞因子介导损伤修复、炎症和纤维化3。由于治疗选择有限，探索IPF的分子病理机制对于制定IPF预防和治疗的新策略具有重要意义。我们小组和其他研究人员4，5 先前的研究已经证实IPF患者和博来霉素（BLM）诱导的IPF小鼠模型中IL-33的释放增加。IL-33在纤维化过程中由上皮和内皮细胞释放并参与巨噬细胞活化，导致成纤维细胞异常增殖，白细胞浸润，最终丧失肺功能5。目前的协议描述了IL-33刺激的间质巨噬细胞（IMs）向肺泡的过继转移，作为研究小鼠模型中IPF发育的一种手段。在这里，从宿主小鼠的肺组织中分离IMs， 体外培养，用IL-33刺激24小时，然后通过气管注射过继转移到受体小鼠的肺泡中。与以前的研究相比，直接收集受刺激的小鼠巨噬细胞并将其过继转移到受体肺泡中会加重肺纤维化的程度，并且可以更清楚地说明刺激因子对纤维化的影响6。本文中描述的技术可以使研究人员探索由潜在细胞因子刺激的巨噬细胞在IPF发展中的功能。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>&#160;DMEM</td>
			<td>Life technologies Biotechnology,USA</td>
			<td>1508012</td>
			<td></td>
		</tr>
		<tr>
			<td>Arterial indwelling needle</td>
			<td>B Braun Melsingen AG,Germany</td>
			<td>21G15G8393</td>
			<td></td>
		</tr>
		<tr>
			<td>BD&#160;Accuri&#160;C6 Plus</td>
			<td>Becton Dickinson,USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bleomycin</td>
			<td>Biotang, USA</td>
			<td>Ab9465</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbon dioxide incubator</td>
			<td>Thermo Forma, USA</td>
			<td>Thermo Forma370</td>
			<td></td>
		</tr>
		<tr>
			<td>CD11b</td>
			<td>R&#38;D Systems,USA</td>
			<td>1124F</td>
			<td></td>
		</tr>
		<tr>
			<td>CD11c</td>
			<td>R&#38;D Systems,USA</td>
			<td>N418</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture dish</td>
			<td>Thermo Forma, USA</td>
			<td>174926</td>
			<td></td>
		</tr>
		<tr>
			<td>Clodronate liposomes&#160;</td>
			<td>Clodronate liposomes,Netherlands</td>
			<td>CI-150-150</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase A</td>
			<td>Sigma-Aldrich, USA</td>
			<td>10103578001</td>
			<td></td>
		</tr>
		<tr>
			<td>F4/80</td>
			<td>R&#38;D Systems,USA</td>
			<td>521204</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon Cell Strainer</td>
			<td>Becton,Dickinson and Company, USA</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Life technologies,USA</td>
			<td>1047571</td>
			<td></td>
		</tr>
		<tr>
			<td>Hematoxylin Eosin&#160;</td>
			<td>Nanjing Jiancheng Technology,China</td>
			<td>06-570</td>
			<td></td>
		</tr>
		<tr>
			<td>LightCycler 480 PCR detection system</td>
			<td>Roche, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Murine recombinant factor IL-33</td>
			<td>Peprotech, USA</td>
			<td>210-33</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon microscope</td>
			<td>Nikon Corporation, Japan</td>
			<td>941185</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin, streptomycin</td>
			<td>Life technologies,USA</td>
			<td>877113</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffer (PBS)</td>
			<td>Guangdong Huankai Microbial Technology ,China</td>
			<td>1535882</td>
			<td></td>
		</tr>
		<tr>
			<td>RBC lysis buffer</td>
			<td>Beyotime Biotechnology Company,China</td>
			<td>C3702</td>
			<td></td>
		</tr>
		<tr>
			<td>RNA Isolater</td>
			<td>Vazyme company,China</td>
			<td>R401-01-AA</td>
			<td>Total RNA extraction reagent</td>
		</tr>
		<tr>
			<td>RWD Inhalation Anesthesia Machine</td>
			<td>Shenzhen Rayward Life Technology ,China</td>
			<td>R500</td>
			<td></td>
		</tr>
		<tr>
			<td>Semi-automatic paraffin slicer</td>
			<td>Leica, Germany</td>
			<td>LeicaRM2245</td>
			<td></td>
		</tr>
		<tr>
			<td>SYBR Premix Ex Taq</td>
			<td>Takara, Japan</td>
			<td>410800</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin 0.25%</td>
			<td>Life Technologies, USA</td>
			<td>1627172</td>
			<td></td>
		</tr>
	</tbody>
</table>

adoptive transfer of il-33-stimulated macrophages into bleomycin-induced mouse models to study their effect on idiopathic pulmonary fibrosis in vivo

早期肺损伤引起的炎症反应是特发性肺纤维化（IPF）发展的重要原因之一，其伴有巨噬细胞和中性粒细胞等炎症细胞的活化，以及包括TNF-α，IL-1β和IL-6在内的炎症因子的释放。已知由活化的肺间质巨噬细胞（IM）响应IL-33刺激引起的早期炎症在IPF的病理过程中起着至关重要的作用。该协议描述了由IL-33刺激的IM的过继转移到小鼠肺部以研究IPF发育。它涉及从宿主小鼠肺部分离和培养原代IM，然后将受刺激的IM过继转移到博来霉素（BLM）诱导的IPF受体小鼠的肺泡中（这些小鼠先前已通过氯膦酸盐脂质体处理耗尽了肺泡巨噬细胞），以及这些小鼠的病理评估。代表性结果表明，IL-33刺激巨噬细胞的过继转移加重了小鼠肺纤维化，表明巨噬细胞过继转移实验的建立是研究IPF病理学的良好技术手段。

图 1 的流程 图总结了此处使用的协议。通过鼻子吸入氯膦酸盐脂质体（图2）用于消耗成年C57BL / 6小鼠的肺巨噬细胞，这产生了良好的受体小鼠模型。从另一只未经治疗的（宿主）小鼠中分离肺IM（图3A，B）并在 体外培养。 用IL-33刺激分离的巨噬细胞24小时，然后气管内滴注到受体小鼠中（图3C</...

Watch this Scientific Journal Video about Adoptive Transfer of IL-33-Stimulated Macrophages into Bleomycin-Induced Mouse Models to Study Their Effect on Idiopathic Pulmonary Fibrosis In Vivo at JoVE.c

Adoptive Transfer of IL-33-Stimulated Macrophages into Bleomycin-Induced Mouse Models to Study Their Effect on Idiopathic Pulmonary Fibrosis In Vivo

该协议描述了肺间质巨噬细胞（IMs）的分离及其在小鼠模型中IL-33刺激肺泡后的过继转移，这可以促进特发性肺纤维化（IPF）的 体内 研究。

将IL-33刺激的巨噬细胞过继转移到博来霉素诱导的小鼠模型中，以研究其对体内特发性肺纤维化的影响

The inflammatory response caused by early lung injury is one of the important causes of the development of idiopathic pulmonary fibrosis (IPF), which is ...

该协议描述了小鼠模型中IL-33测定后肺IMS的分离和过继转移，这可以促进特发性肺纤维化的体内研究。该技术使研究人员能够探索巨噬细胞的功能，由传统细胞因子模拟IPF发展。首先，从冰箱中取出一瓶氯膦酸盐脂质体。让它在室温下加热 30 分钟，然后倒置几次以确保混合均匀。使用带有无菌吸头的移液管吸出60微升氯膦酸盐脂质体。将对照和药物滴入麻醉小鼠的鼻腔中。每次滴后，确保小鼠完全吸入药物并均匀呼吸。在受体小鼠中肺泡巨噬细胞耗尽后，首先用75%酒精和碘对麻醉宿主小鼠的皮肤进行消毒。用剪刀剪开皮肤，露出心肺组织。在配备20号针头的注射器中吸出10毫升PBS，并将针尖插入小鼠的右心房。然后用手术剪刀切割小鼠的下腔静脉，并以每分钟10至20毫升的恒定速度手动用PBS灌注小鼠，直到肺组织变白。此外，切除肺组织并将其转移到培养皿中的冰冷PBS中。将肺组织切成碎片，加入15毫升含有1%胶原酶A的DMEM培养基以分离巨噬细胞，然后在37摄氏度的摇床上以100 RPM孵育30分钟。通过10毫升注射器吸出肺组织悬浮液约20次以使其精细。通过40微米细胞过滤器过滤悬浮液。将滤液以400G离心10分钟。弃去上清液后，用三毫升细胞裂解缓冲液在冰上裂解红血两至三分钟。以 150 G 离心五分钟，然后将细胞沉淀重悬于 10 毫升 DMEM 中。然后使用血细胞计数器计数细胞。将细胞接种到10厘米的贴壁细胞培养皿中，并在37摄氏度和5%二氧化碳下孵育一小时，以将肺间质巨噬细胞粘附在培养皿上。一小时后，吸出上清液和浮动细胞。加入 10 毫升新鲜、完整的培养基，孵育超过 8 小时或过夜。为了刺激分离的间质巨噬细胞，用完整的白细胞介素33替换用过的培养基。还通过添加PBS代替白细胞介素33来制备对照板。将板在 37 摄氏度和 5% 二氧化碳下孵育 24 小时。要解离肺间质巨噬细胞，请用一毫升0.25%胰蛋白酶治疗五分钟。然后加入三毫升新鲜培养完全培养基淬灭消化并离心细胞悬液收获肺巨噬细胞。计数细胞后，将细胞重悬于PBS中至终浓度为每50微升10至第五个细胞的五倍。为了开始转移间质巨噬细胞，将麻醉受体小鼠的四肢固定在带有医用胶带的垂直板上，并使用棉签轻轻地将小鼠的舌头拉到一侧。打开插管灯后，将其照在小鼠的喉咙上，以查看气管，气管应靠近舌根。使用 22 号插管灯和 0.4 毫米直径导丝将留置针套管插入气管。要完成插管，请取下导丝并将套管推入小鼠气管。现在，当看到套管进入气管时，通过气管将50微升细胞悬液注射到受体小鼠中。24 小时后，通过气管给予博来霉素以诱导特发性肺纤维化。同时向对照组施用等体积的盐水。21 天后，通过评估纤维化标志物的表达和 Ashcroft 评分来确定肺纤维化的严重程度。HE染色显示，与对照组相比，白细胞介素33刺激巨噬细胞的过继转移加剧了博来霉素刺激小鼠的肺组织破坏程度并增加了成纤维细胞聚集。阿什克罗夫特评分的增加也说明了肺纤维化的程度。与BLM处理的野生型小鼠相比，含有过继转移白细胞介素33刺激的间质巨噬细胞并用博来霉素处理的受体小鼠肺组织中α SMA和纤连蛋白的mRNA表达水平更高。应立即用一毫米注射器将500微升空气输入肺部，以确保套管中的细胞悬浮液能够完全进入肺组织。

Research

JoVE Journal

Immunology and Infection

Adoptive Transfer of IL-33-Stimulated Macrophages into Bleomycin-Induced Mouse Models to Study Their Effect on Idiopathic Pulmonary Fibrosis In Vivo

Experimental Metastasis and CTL Adoptive Transfer Immunotherapy Mouse Model

实验转移和CTL过继转移免疫小鼠模型

Experimental metastasis mouse model is a simple and yet physiologically relevant metastasis model. The tumor cells are injected intravenously (i.v) into ...

科研

免疫与感染

Overcoming Unresponsiveness in Experimental Autoimmune Encephalomyelitis (EAE) Resistant Mouse Strains by Adoptive Transfer and Antigenic Challenge

Overcoming Unresponsiveness in Experimental Autoimmune Encephalomyelitis EAE Resistant Mouse Strains by Adoptive Transfer and Antigenic Challenge

在实验性自身免疫性脑脊髓炎（EAE）抗性品系小鼠继转移和抗原挑战克服反应迟钝

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the central nervous system (CNS) and has been used as an animal model for ...

Isolation, Purification and Labeling of Mouse Bone Marrow Neutrophils for Functional Studies and Adoptive Transfer Experiments

功能研究和过继转移实验小鼠骨髓嗜中性粒细胞的分离，纯化及贴标

Neutrophils are critical effector cells of the innate immune system. They are rapidly recruited at sites of acute inflammation and exert protective or ...

Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

使用<em&gt;福氏痢疾杆菌</em&gt;研究自噬，细胞骨架相互作用

Shigella flexneri is an intracellular pathogen that can escape from phagosomes to reach the cytosol, and polymerize the host actin cytoskeleton to promote ...

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

诱导小鼠肠道炎症由效应CD4的过继转移<sup&gt; +</sup&gt; CD45RB<sup&gt;高</sup&gt; T细胞免疫缺陷小鼠

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...

Generation of Lymphocytic Microparticles and Detection of their Proapoptotic Effect on Airway Epithelial Cells

淋巴细胞微粒和检测其促凋亡作用的气道上皮细胞产生

Interest in the biological roles of cell membrane&#8211;derived vesicles in cell&#8211;cell communication has increased in recent years. Microparticles ...

Intranasal Administration of Recombinant Influenza Vaccines in Chimeric Mouse Models to Study Mucosal Immunity

重组流感疫苗的嵌合体小鼠模型鼻腔给药研究黏膜免疫

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the ...

Intra-tracheal Administration of Haemophilus influenzae in Mouse Models to Study Airway Inflammation

Intra-tracheal Administration of Haemophilus influenzae in Mouse Models to Study Airway Inflammation

的气管内管理<em&gt;流感嗜血杆菌</em&gt;在小鼠模型来研究气道炎症

Here, we describe a detailed procedure to efficiently and directly deliver Haemophilus influenzae into the lower respiratory tracts of mice. We ...

Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages

Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages

嗜肺军团菌外膜囊泡：隔离及对巨噬细胞促炎潜力分析

Bacteria are able to secrete a variety of molecules via various secretory systems. Besides the secretion of molecules into the extracellular space or ...

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

IL-8瞬时转化的小鼠模型<em&gt;体内</em&gt;长期监测炎症反应

Airway inflammation is often associated with bacterial infections and represents a major determinant of lung disease. The in vivo determination of the ...