Name: bone marrow transplantation procedures in mice to study clonal hematopoiesis
Uploaded: 2021-05-26T13:00:00
Description: Watch this Scientific Journal Video about Bone Marrow Transplantation Procedures in Mice to Study Clonal Hematopoiesis at JoVE.com

这项工作得到了美国国家卫生研究院对K.Walsh（HL131006，HL138014和HL132564）的资助，对S.Sano（HL152174）、美国心脏协会对M.A.Evans（20POST35210098）的赠款以及日本心脏基金会对小川的赠款的支持。

所有涉及动物学科的程序都已得到弗吉尼亚大学动物护理和使用委员会 （IACUC） 的批准。
1. 在先决条件之前
<ol><li>在辐照前将受助小鼠置于抗生素补充水（5m 磺酰太极，0.86 mM 三氯苯丙酮）~24h。这是预防感染所必需的，因为免疫系统在照射后会受到抑制，并在辐照后维持2周。此时，用营养/水化凝胶补充小鼠，以鼓励喂养，防止辐照后体重减轻和脱水。</li>
</ol><p class="jove_c...

<ol>
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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=The+impact+of+aging+on+primate+hematopoiesis+as+interrogated+by+clonal+tracking.">The impact of aging on primate hematopoiesis as interrogated by clonal tracking.</a> Blood. 131 (11), 1195-1205 (2018).</li><li>Sano, 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=CRISPR-mediated+gene+editing+to+assess+the+roles+of+Tet2+and+Dnmt3a+in+clonal+hematopoiesis+and+cardiovascular+disease.">CRISPR-mediated gene editing to assess the roles of Tet2 and Dnmt3a in clonal hematopoiesis and cardiovascular disease.</a> Circulation Research. 123 (3), 335-341 (2018).</li><li>Stachura, D. 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=Clonal+analysis+of+hematopoietic+progenitor+cells+in+the+zebrafish.">Clonal analysis of hematopoietic progenitor cells in the zebrafish.</a> Blood. 118 (5), 1274-1282 (2011).</li><li>Gyurkocza, B., Sandmaier, B. 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Z., 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=Optimal+protocol+for+total+body+irradiation+for+allogeneic+bone+marrow+transplantation+in+mice.">Optimal protocol for total body irradiation for allogeneic bone marrow transplantation in mice.</a> Bone Marrow Transplantation. 30 (12), 843-849 (2002).</li><li>Koch, A., 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=Establishment+of+early+endpoints+in+mouse+total-body+irradiation+model.">Establishment of early endpoints in mouse total-body irradiation model.</a> PLOS One. 11 (8), 0161079 (2016).</li><li>Ismaiel, A., Dumitra&#351;cu, D. 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对于克隆造血症的研究，我们描述了BMT的三种方法：具有全身辐照的BMT、带部分屏蔽的辐照BMT，以及一种不太常用的BMT方法，不涉及预调理（采用BMT）。这些方法已用于评估克隆血吸管对心血管疾病的影响。研究人员可以相应地修改这些方法，以适应他们研究的具体目的。
克隆造血模型 克隆造血是突变造血细胞与野生类型细胞竞争并随着时间的推移获得克隆?...

克隆造血症 （CH） 是一种在老年人中经常观察到的疾病，并且由于携带基因突变1的扩大造血干细胞和祖细胞 （HSPC） 克隆而发生。有人建议，到50岁时，大多数个体在每一个HSPC2中平均会获得5个外向突变，但大多数这些突变对个体的影响很小或根本没有。然而，如果这些突变中偶然的一个给HSPC带来竞争优势，例如通过促进它的增殖、自我更新、生存或这些突变的某种组合——这可能导致突变克隆相对于其他HSC的优先扩展。因此，随着突变的HSPC产生成熟的血细胞，突变将越来越多地通过造血系统传播，导致周围血液中突变细胞的明显数量。虽然数十种不同候选驱动基因的突变与造血系统中的克隆事件有关，但其中，DNA甲基转移酶3α（DNMT3A）和11个转移2（TET2）的突变是最普遍的3。一些流行病学研究发现，携带这些基因突变的个体患心血管疾病（CVD）、中风和全因死亡率的风险要高得多。虽然这些研究已经确定CH与心乳炎和中风发病率增加之间存在关联，但我们不知道这种关系是因果关系还是与衰老过程共享的表皮。为了更好地了解这种关联，需要适当的动物模型来正确地回顾CH的人类状况。
我们小组和其他使用斑马鱼、老鼠和非人类灵长类动物8、9、10、11、12、13、14建立了若干CH动物模型。这些模型通常使用造血重组方法移植转基因细胞，有时使用Cre-lox重组或CRISPR系统。这种方法允许分析造血细胞中的特定基因突变，以评估它如何促进疾病发展。此外，这些模型通常使用致癌细胞或报告细胞来区分突变细胞与正常或野生细胞的影响。在许多情况下，需要预调理方案才能成功移植供体造血干细胞。
目前，骨髓移植到受体小鼠可分为两大类：1）骨髓移植和2）无条件移植。骨髓调节可以通过两种方法之一，即全身照射（TBI）或化疗15实现。TBI是通过让接受者接受致命剂量的伽马或X射线照射，在快速分裂的细胞内产生DNA断裂或交叉链接，使它们无法弥补16。布苏尔凡和环磷酰胺是两种常用的化疗药物，它们破坏造血利基，并同样对快速分裂的细胞造成DNA损伤。骨髓疏松预制的最终结果是造血细胞凋亡，从而破坏了受体的造血系统。这一战略不仅使捐赠者HSPC能够成功获得，而且可以通过抑制接受者的免疫系统来防止移植排斥。然而，骨髓调节有严重的副作用，如损害组织和器官及其居民免疫细胞，以及破坏原生骨髓利基17。因此，提出了克服这些不良副作用的替代方法，特别是在对利益机关的损害方面。这些方法包括受体小鼠的屏蔽辐照和采用BMT对非条件小鼠9，17。通过放置铅屏障保护胸腔、腹腔、头部或其他区域免受辐照，使感兴趣的组织免受辐照的破坏性影响，并维持其常驻免疫细胞群。另一方面，HSPC对无条件小鼠的采用BMT具有额外的优势，因为它保留了原生的造血利基。在本手稿中，我们描述了在小鼠中移植几种方案后HSPC移植的协议和结果，特别是将HSPC输送给TBI小鼠、部分不受辐照的老鼠和非条件小鼠。总体目标是帮助研究人员了解每种方法的不同生理影响，以及它们如何影响CH和心血管疾病设置中的实验结果。

<table>
	<tbody>
		<tr>
			<td>0.5ml microcentrifuge</td>
			<td>Fisher Scientific</td>
			<td>05-408-121</td>
			<td>general supply</td>
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			<td>1.5ml microcentrifuge</td>
			<td>Fisher Scientific</td>
			<td>05-408-129</td>
			<td>general supply</td>
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			<td>1/2 cc LO-DOSE INSULIN SYRINGE</td>
			<td>EXELINT</td>
			<td>26028</td>
			<td>general supply</td>
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			<td>Absolute Ethanol (200 prfof)</td>
			<td>Fisher chemical</td>
			<td>200559</td>
			<td>general supply</td>
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			<td>BD 1mL Tuberculin Syringes 25G 5/8 Inch Needle</td>
			<td>Becton Dickinson</td>
			<td>309626</td>
			<td>general supply</td>
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			<td>BD PrecisionGlide Needle 18G (1.22mm X 25mm)</td>
			<td>Becton Dickinson</td>
			<td>395195</td>
			<td>general supply</td>
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			<td>Cesium-137 Irradiator</td>
			<td>J. L. Shepherd&#160;</td>
			<td>Mark IV</td>
			<td>equipment</td>
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			<td>DietGel 76A</td>
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			<td>70-01-5022</td>
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			<td>Life Sciences</td>
			<td>353003</td>
			<td>general supply</td>
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			<td>Falcon 50 mL Conical Centrifuge Tubes</td>
			<td>Fisher Scientific</td>
			<td>352098</td>
			<td>general supply</td>
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			<td>Fisherbrand sterile cell strainers, 70 &#956;m</td>
			<td>Fisher Scientific</td>
			<td>22363548</td>
			<td>general supply</td>
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			<td>Graefe Forceps</td>
			<td>Fine Science Tools</td>
			<td>11051-10</td>
			<td>general supply</td>
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			<td>Hardened Fine Scissors</td>
			<td>Fine Science Tools</td>
			<td>14090-09</td>
			<td>general supply</td>
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			<td>Isothesia (Isoflurane) solution</td>
			<td>Henry Schein</td>
			<td>29404</td>
			<td>Solution</td>
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			<td>Ketamine</td>
			<td>Zoetis</td>
			<td>043-304</td>
			<td>injection</td>
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			<td>Kimwipes Delicate Task Wipers</td>
			<td>Kimtech Science</td>
			<td>KCC34155</td>
			<td>general supply</td>
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			<td>PBS pH7.4 (1X)</td>
			<td>Gibco</td>
			<td>10010023</td>
			<td>Solution</td>
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			<td>RadDisk &#8211; Rodent Irradiator Disk</td>
			<td>Braintree Scientific</td>
			<td>IRD-P M</td>
			<td>general supply</td>
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			<td>RPMI Medium 1640 (1X)</td>
			<td>Gibco</td>
			<td>11875-093</td>
			<td>Medium</td>
		</tr>
		<tr>
			<td>Sulfamethoxazole and Trimethoprim</td>
			<td>TEVA</td>
			<td>0703-9526-01</td>
			<td>injection</td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Akorn</td>
			<td>139-236</td>
			<td>injection</td>
		</tr>
		<tr>
			<td>X-ray irradiator</td>
			<td>Rad source</td>
			<td>RS-2000</td>
			<td>equipment</td>
		</tr>
	</tbody>
</table>

bone marrow transplantation procedures in mice to study clonal hematopoiesis

克隆造血是一种普遍的年龄相关疾病，是造血干细胞和祖细胞（HSPCs）体细胞突变积累的结果。驱动基因的突变，赋予细胞的适应性，可以导致扩大HSPC克隆的发展，越来越多地导致后代白细胞怀有体细胞突变。由于克隆造血症与心脏病、中风和死亡率有关，因此开发模拟这些过程的实验系统是理解这一新危险因素的机制的关键。骨髓移植程序涉及小鼠骨髓移植调节，如全身照射（TBI），通常用于研究免疫细胞在心血管疾病中的作用。然而，同时损害骨髓利基和其他感兴趣的部位，如心脏和大脑，是不可避免的与这些程序。因此，我们的实验室已经开发出两种替代方法，以尽量减少或避免TBI可能造成的副作用：1） 骨髓移植与辐照屏蔽和 2） 采用BMT到非条件小鼠。在屏蔽器官中，保留局部环境，以便分析克隆造血，同时居民免疫细胞的功能不受干扰。相比之下，对无条件小鼠采用BMT具有额外的优势，即保留器官的局部环境和造血利基。在这里，我们比较了三种不同的造血细胞重组方法，并讨论了它们在心血管疾病中克隆造血症研究的长处和局限性。

为了比较三种BMT/预调理方法对供体细胞移植的影响，通过BMT后1个月的流动细胞测量分析外周血液和心脏组织中供体细胞的分数。分离细胞被染色为特定的白细胞标记物，以识别白细胞的不同子集。在这些实验中，野生型C57BL/6（CD45.2）供体骨髓细胞被输送到WT B6。SJL-Ptprc一个Prpcb/ BoyJ （CD45.1） 受体小鼠，以区分受体细胞中的供体细胞和受体...

Watch this Scientific Journal Video about Bone Marrow Transplantation Procedures in Mice to Study Clonal Hematopoiesis at JoVE.com

Bone Marrow Transplantation Procedures in Mice to Study Clonal Hematopoiesis

我们描述了三种骨髓移植方法（BMT）：全身辐照的BMT、屏蔽辐照的BMT和无预调理（采用BMT）的BMT方法，用于研究小鼠模型中的克隆造血。

小鼠骨髓移植程序研究克隆造血症

Clonal hematopoiesis is a prevalent age-associated condition that results from the accumulation of somatic mutations in hematopoietic stem and progenitor ...

我们的协议将帮助研究人员了解三种不同的骨髓移植方法的生理影响，以及它们如何影响克隆造血症设置中的实验结果。全身辐射骨髓移植会对心血管器官产生负面影响，改变疾病发病机理。因此，我们的实验室已经开发了两种替代方法，以尽量减少或避免可能的副作用。演示程序将是恩比帕克一个研究生，梅根埃文斯博士后研究员，无论是从我的实验室为胸腔和腹部屏蔽，把可调托盘和X射线辐照器在正确的距离，以实现统一的辐照。并将麻醉小鼠放在平底板上，在超平位置相互倒置。用胶带将爪子固定在板上，并将引头屏蔽放置，使下端与西菲斯特纳姆骨对齐，铅盾的上端位于胸腺附近。屏蔽后，将小鼠放入辐照器中，使动物暴露在两个5.5灰色的辐照下，间隔4至24小时。为了遮挡头部，小心地将鼠标的四只爪子贴在腹部。将鼠标放入锥形约束器中，将约束器滑入铅盾内的插槽中，使鼠标的头部和耳朵完全覆盖，使小鼠身体的其他部位暴露在辐射之下。屏蔽后，将小鼠放入辐照器中，使动物暴露在4至24小时间隔内分离的5.5个灰色辐射分数。每次放射治疗后，将麻醉小鼠放在加热垫上的笼子里进行监测，直到完全康复。为了隔离骨骼，用70%乙醇对供体小鼠的皮肤进行消毒，并在肋骨笼下方做一个小的横向皮肤切口。将皮肤紧紧地抱在切口的两侧，朝头部和脚部朝相反的方向撕裂，从所有四肢剥落皮肤。切开肩膀和肘关节，使用实验室擦拭从腐殖质中去除附着的肌肉和结缔组织。小心地脱位股骨和臀部骨骼之间的关节。并用钝剪刀沿着股骨头切开腿。切开膝盖关节，分离股骨和头骨。并使用实验室湿巾小心地从骨骼中取出附着的肌肉和结缔组织。然后将同一基因型小鼠的骨骼拉入含有冰上20毫升冰冷无菌PBS的50毫升圆锥管中。要隔离生物安全类二柜中的骨髓细胞，请使用 18 号针头在无菌 500 微升微管底部打一个小孔。并将管子放入含有100微升冰冷无菌PBS的1.5毫升微离心管中。当所有管子都准备好后，从含有分离骨骼的管中取出 PBS，并将骨骼转移到无菌的 100 毫米细胞培养盘上。使用细钳和小剪刀小心地从每根骨头的末端去除表皮，并将多达六块骨头放入每个500微滑管中。当所有的骨头都被切割后，通过离心提取骨髓细胞。如果所有骨髓内容都已去除，骨骼应呈白色和半透明，1.5毫升微离心管底部有一个相对较大的红色颗粒。对于骨髓细胞移植，稀释血清中分离的骨髓细胞，将200微升的细胞悬浮物加载到每只小鼠0.5毫升胰岛素注射器中注射。在确认对踏板反射缺乏反应后，慢慢地将整个细胞体积注入每个麻醉接受动物的复古轨道静脉。然后将一滴丙丙卡因放在眼睛上以缓解疼痛。并允许鼠标在被监视时恢复知觉。比较三种BMT预置方法对供体细胞移植的影响。在BMT后一个月，通过流动细胞学对周围血液和心脏组织中供体细胞的分数进行了分析。在这项具有代表性的分析中，在接受全身照射的受体小鼠的外周血液中，单核细胞、嗜中性粒细胞和B细胞基本上被消融，取而代之的是供体骨髓衍生细胞的后代。此外，常驻接受者心脏单核细胞和中性粒细胞种群几乎完全被供体衍生细胞所取代。在部分屏蔽的辐照组中，捐赠者衍生的心脏免疫细胞置换量不大。与接受全身照射的小鼠相比，受助小鼠骨髓细胞在屏蔽区域内的外周血液重组水平较低。在没有BMT预制条件的组中，在BMT之后的四周内，接受者小鼠的周围血液和心脏中可检测出供体衍生细胞。此外，Tet2缺乏的供体细胞随着时间的推移逐渐扩大。相比之下，受体小鼠与野生类型的供体细胞一起生长，显示出供体细胞的克隆扩张最小。他们优化这些实验模型将使对克隆造血驱动基因进行更严格的研究，这些基因有助于所有导致死亡率，如心代谢疾病和癌症。我们希望，我们的协议将允许研究人员研究克隆造血症，以研究它如何有助于心血管疾病和其他疾病状态。

Research

JoVE Journal

Immunology and Infection

Toxoplasma gondii Cyst Wall Formation in Activated Bone Marrow-derived Macrophages and Bradyzoite Conditions

Toxoplasma gondii Cyst Wall Formation in Activated Bone Marrow-derived Macrophages and Bradyzoite Conditions

弓形虫囊肿壁形成激活骨髓衍生的巨噬细胞和Bradyzoite条件

Toxoplasma gondii is an obligate intracellular parasite that can invade any nucleated cell of warm-blooded animals. During infection, T. gondii ...

科研

免疫与感染

Differentiating Functional Roles of Gene Expression from Immune and Non-immune Cells in Mouse Colitis by Bone Marrow Transplantation

区分功能基因表达的作用，通过骨髓移植免疫和非免疫细胞在小鼠结肠炎

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice ...

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 ...

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

长期慢性<em&gt;铜绿假单胞菌</em&gt;在呼吸道感染小鼠

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model ...

Transplantation of Tail Skin to Study Allogeneic CD4 T Cell Responses in Mice

尾部皮肤来研究异基因CD4 + T细胞应答的小鼠移植

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and ...

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, ...

Retroviral Transduction of Bone Marrow Progenitor Cells to Generate T-cell Receptor Retrogenic Mice

骨髓祖细胞的逆转录病毒转导到生成T细胞受体Retrogenic小鼠

T cell receptor (TCR) signaling is essential in the development and differentiation of T cells in the thymus and periphery, respectively.  The vast array ...