Name: intra-peritoneal transplantation for generating acute myeloid leukemia in mice
Uploaded: 2023-01-06T14:10:00
Description: Watch this Scientific Journal Video about Intra-Peritoneal Transplantation for Generating Acute Myeloid Leukemia in Mice at JoVE.com

作者感谢哈克研究所的流式细胞术核心设施和宾夕法尼亚州立大学兽医和生物医学科学系动物诊断实验室的组织病理学核心设施提供及时的技术支持。这项工作得到了美国癌症研究所（KSP），宾夕法尼亚州立大学农业科学学院，宾夕法尼亚州癌症研究所，USDA-NIFA项目4771，KSP和RFP的入藏号00000005的资助。

所有实验均由宾夕法尼亚州立大学机构动物护理和使用委员会预先批准。
1. 缓冲液和试剂的制备
<ol><li>准备氨苄青霉素补充（AP）LB琼脂平板（无菌10厘米平板）。为此，将 10 克 LB 肉汤与琼脂溶解在 400 mL 蒸馏水中，搅拌，使体积达到 500 mL。通过高压灭菌对溶液进行灭菌，然后让溶液冷却，向溶液中加入0.5mL氨苄西林（库存：150mg / mL），摇晃混合。立即将18mL溶?...

<ol>
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这些上述研究提供了支持性证据，证明Lin- 细胞的移植在1°鼠AML的产生中与LSK细胞相当。此外，目前的数据还表明，与静脉内（或r.o.）注射相比，腹腔注射是建立鼠AML的一种有效便捷的方法。
除LSK细胞外，据报道，其他群体（如粒细胞-单核细胞祖细胞（GMP）、淋巴总祖细胞（CLP）和髓系总祖细胞（CMP））在产生1° MLL-AF9诱导的AML时被取代为供体细胞，具有不同的孵育...

急性髓系白血病（AML）是一种病因多样的血液系统恶性肿瘤，预后不良1。AML动物模型的产生为理解其复杂的变异和病理生物学奠定了基础，以努力发现新的疗法2。小鼠的白血病发生涉及表达融合癌蛋白的供体细胞的移植，包括涉及混合谱系白血病（MLL）基因的融合，以有效诱导AML，以模拟人类的疾病3。在MLL基因相关AML4的移植中，已经报道了供体细胞的各种细胞起源，对导致疾病起源的细胞知之甚少。
已经开发了多种途径用于小鼠移植;与将突变供体细胞直接引入骨髓5 的股内注射不同，利用静脉窦丛、尾静脉和颈静脉的静脉注射已被广泛用于生成鼠 AML 模型6，7，8，9。在眶后（r.o.）注射的情况下，各种固有的缺点，如体积限制、高技术要求、重复尝试或错误的机会很少以及潜在的眼部损伤，一直是主要的绊脚石，有限的或没有可行的替代方案7。除了局部损伤外，尾静脉注射还可能存在类似的问题;为了促进手术，小鼠通常需要加热以扩张尾静脉10。如果没有额外的光源，也很难找到尾静脉，特别是在小鼠的C57BL / 6品系中。对于颈静脉注射，研究人员需要足够的培训来定位静脉并限制可能的并发症。此外，静脉窦和颈静脉注射都需要在麻醉下进行，这增加了另一个层次的复杂性。因此，探索新的移植途径以促进AML小鼠模型的建立是很诱人的。
腹膜内（ip）注射通常用于施用药物，染料和麻醉剂11，12，13，14，15;它还被用于引入异位造血的造血细胞16，并在各种小鼠模型中移植骨髓来源的间充质干细胞17，18，19，20，21。然而，它很少用于建立小鼠的造血恶性肿瘤，特别是研究AML疾病的进展。
除了比较谱系阴性（Lin-）和Lin-Sca-1 + c-Kit+（LSK）群体作为供体细胞的移植效率外，本研究还描述了腹腔注射在AML小鼠模型生成中的可行性。这些发现为生成AML和相关髓系白血病的实验模型提供了一种简单有效的方法。这种方法有可能进一步了解疾病机制，并提供一个相对容易的模型来测试实验性疗法。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
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			<td>a-Select competent cells&#160;</td>
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			<td>Gibco</td>
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intra-peritoneal transplantation for generating acute myeloid leukemia in mice

治疗急性髓系白血病（AML）和涉及持续性白血病干细胞（LSCs）的相关复发的新疗法的需求尚未得到满足。一种实验性AML啮齿动物模型测试基于通过眶后注射在受体小鼠中成功移植这些细胞的疗法，充满了挑战。本研究的目的是开发一种简单、可靠和一致的方法，使用腹膜内途径生成强大的 AML 小鼠模型。在本协议中，骨髓细胞用表达人MLL-AF9融合癌蛋白的逆转录病毒转导。测试了谱系阴性（Lin-）和Lin-Sca-1 + c-Kit+（LSK）群体作为供体LSC在原发性AML发展中的效率，并采用腹膜内注射作为产生AML的新方法。在连续移植中对腹膜内和眶后注射进行比较，以比较和对比两种方法。用人MLL-AF9病毒转导的Lin细胞和LSK细胞都很好地移植在受体的骨髓和脾脏中，导致全面的AML。连续移植时腹膜内注射供体细胞在受者中建立AML，并通过流式细胞术、qPCR和组织学分析在受者的血液、骨髓、脾脏和肝脏中检测到AML细胞浸润。因此，腹膜内注射是一种使用供体白血病细胞连续移植诱导AML的有效方法。

使用r.o.和ip.移植途径的小鼠AML细胞移植效率的比较 以前，在用MLL-AF9转导的LSK细胞进行眶后移植的受体小鼠中报告了1°AML的建立，并且通过连续移植证明了1°AML细胞的可移植性30。本研究首次评估使用骨髓林细胞 进行移植的可能性。异常白细胞增多（图2A）的存在和骨髓和脾脏中白血病细胞（CD45.1+）浸润增加（<strong class="xfig...

Watch this Scientific Journal Video about Intra-Peritoneal Transplantation for Generating Acute Myeloid Leukemia in Mice at JoVE.com

Intra-Peritoneal Transplantation for Generating Acute Myeloid Leukemia in Mice

在这里，腹膜内注射白血病细胞用于在小鼠中建立和繁殖急性髓性白血病（AML）。这种新方法在AML细胞的连续移植中是有效的，并且可以作为那些可能在小鼠静脉注射中遇到困难和不一致的人的替代方案。

腹膜内移植在小鼠中产生急性髓系白血病

There is an unmet need for novel therapies to treat acute myeloid leukemia (AML) and the associated relapse that involves persistent leukemia stem cells ...

该方案很重要，因为从同一供体小鼠分离的lin阴性细胞和SK细胞在MLL-AF9诱导的AML的产生中没有明显的差异。此外，白血病细胞的IP注射可以成功开发AML的小鼠模型。与传统的移植方法相比，这种技术更方便，更便宜。首先，用8%乙醇对8至10周龄的CD45.1雌性C57BL / 6J小鼠进行灭菌。将鼠标放在泡沫塑料板上的无菌手术垫上，然后将腿穿过鼠标爪垫。在中线切开腹腔上方的皮肤，并用锋利的无菌剪刀将皮下空间扩大到后腿。将切口从腹部中线向下延伸到脚踝，并用锋利的剪刀刀片扩大后腿下方的皮下空间。用锋利的剪刀剪断跟腱。用带齿的镊子握住肌腱，切开附着在股骨上的另一端以去除腓肠肌。用锋利的剪刀剪断附着在膝盖上的股四头肌肌腱。握住肌腱并切开附着在股骨上的肌肉头以去除腓肠肌。切断连接到胫骨末端的股骨周围的其他肌肉。接下来，用锋利的剪刀剪断脚踝，确保胫骨保持完整。握住股骨远端，用锋利的剪刀剪断髋关节，确保股骨头保持完整。将胫骨和股骨转移到 15 毫升管中的流动缓冲液中。用手打破膝盖来分开胫骨和股骨。切除髌骨、软骨和股骨髁，露出胫骨平台和股骨远端。使用无菌纱布去除肌肉，并将骨骼浸泡在流动缓冲液中。切开股骨颈，并使用带有23号针头的10毫升注射器从股骨两端用流动缓冲液冲洗骨髓细胞。接下来，切开胫踝并用来自胫骨两端的流动缓冲液冲洗骨髓细胞。通过使用带有18号针头的10毫升注射器上下移液来分散细胞。将单细胞悬液在4摄氏度和400G下离心三分钟。弃去上清液并将细胞重悬于五毫升红细胞或红细胞裂解缓冲液中以裂解红细胞三分钟。加入五毫升流动缓冲液以停止裂解。然后离心细胞悬液并将沉淀重悬于五毫升流动缓冲液中。将70微米的细胞过滤器放在50毫升的管上，并将悬浮液通过过滤器以收集细胞。在圆底聚丙烯管中，用流动缓冲液将细胞浓度调节至每毫升10至8倍。根据制造商的说明，使用小鼠造血细胞分离试剂盒选择谱系阴性细胞。完成后，离心分选的造血干细胞和未分选的谱系阴性细胞，并在反还原剂包衣的培养皿中重悬于三毫升 2X 细胞因子、IMDM 培养基和三毫升病毒上清液中。将培养皿在 37 摄氏度的加湿 5% 二氧化碳培养箱中孵育 6 或 24 小时。转导后，通过离心收获细胞。如果需要，使用胰蛋白酶收集附着在培养皿底部的细胞。弃去上清液并将沉淀重悬于预热的PBS中。用27号半针将细胞眶后或腹膜内注射到主要受体小鼠中。每天监控鼠标。一个月后，每周通过眶后出血采集血液，通过评估 HEMAVET 的全血细胞计数来监测白细胞增多。用拇指和食指撑起眼睛，然后用无菌血细胞比容毛细管穿过内眦穿透静脉窦丛。将20至25微升血液收集到EDTA采血管中并关闭眼睑以止血。将一滴硫酸庆大霉素眼用溶液涂抹在眼睛上。当白细胞达到每微升 10 到 4 个细胞的四倍时，通过用流动缓冲液冲洗股骨和胫骨来分离骨髓细胞，然后如前所述进行红细胞裂解。接下来，为了收获脾细胞，将鼠标放在泡沫塑料板上的无菌手术垫上，并将腿穿过鼠标爪垫。用70%乙醇对小鼠进行消毒。用锋利的无菌剪刀切开中线的皮肤和肌肉，露出腹腔。分离脾脏并将其放入15毫升管中的流动缓冲液中。在装有三毫升流动缓冲液的 6 厘米培养皿中通过 70 微米过滤器将脾脏啮合。将细胞从培养皿转移到15毫升管中，然后离心单细胞悬液，弃去上清液，并将沉淀重悬于5毫升红细胞裂解缓冲液中3分钟。加入五毫升流动缓冲液以停止裂解并离心细胞悬液。将沉淀重悬于5毫升流动缓冲液中，混合并通过70微米细胞过滤器，将细胞收集到50毫升管中。通过用FITC偶联的抗小鼠CD45.1抗体染色脾细胞和骨髓细胞并在流式细胞仪上进行检测来鉴定原发性急性髓系白血病或AML细胞。首先在FSC-A FSC-H和FSC-A SSC-A上对细胞进行门控以获得单线态。通过将其与未染色的细胞进行比较，在 FL1 图上对 CD45.1 阳性群体进行门控。对于二次移植，将来自PBS中主要眶后受者的CD45.1 AML脾细胞重悬，并将其眶后注射到CD45.2雄性小鼠中。骨髓和脾脏中白细胞增多和白血病细胞浸润增加的存在支持了使用骨髓谱系阴性细胞产生原发性 AML 的可行性。在用LSK或谱系阴性细胞翻译的主要受者中没有观察到显着差异。AML细胞通过混合谱系白血病或MLL-AF9转导谱系阴性细胞在主要受体的腹腔中扩散。这些结果还证实了通过腹膜内注射白细胞增多形式的MLL-AF9转导骨髓谱系阴性细胞以及受体小鼠骨髓和脾脏中AML细胞的存在来建立原发性AML。然而，尽管供体细胞数量相同，但通过腹膜内注射进行初次移植比通过眶后注射需要更长的时间才能产生AML。次要受者在移植后不到一个月内表现出白细胞增多和明显的肝脾肿大。在外周血、骨髓和脾脏以及腹膜腔中也检测到AML细胞。腹膜内注射8乘以10到5个AML细胞，实现了与8乘以10到5个AML的眶后注射相当的植入。三级受体小鼠急性表现出AML体征，包括白细胞增多和肝脾肿大，血液，骨髓和脾脏中存在白血病细胞，股骨和脾脏的组织学观察进一步证明了白血病细胞的浸润。与二级和三级移植相比，三级移植的进展比二次移植快得多。对于当前模型，不需要对 LSK 单元格进行排序。林阴性细胞可以用AML病毒转导6小时或24小时。它不会影响模型建立的效率。

Research

JoVE Journal

Cancer Research

A Detailed Protocol for Characterizing the Murine C1498 Cell Line and its Associated Leukemia Mouse Model

详细的定性C1498小鼠细胞系及其关联白血病小鼠模型协议

The intravenous injection of C1498 cells into syngeneic or congenic mice has been performed since 1941. These injections result in the development of ...

科研

癌症研究

Comprehensive Protocol to Sample and Process Bone Marrow for Measuring Measurable Residual Disease and Leukemic Stem Cells in Acute Myeloid Leukemia

测定急性髓系白血病可测残留病和白血病干细胞的骨髓标本和过程综合协议

Response criteria in acute myeloid leukemia (AML) has recently been re-established, with morphologic examination utilized to determine whether patients ...

Preparation of Primary Acute Lymphoblastic Leukemia Cells in Different Cell Cycle Phases by Centrifugal Elutriation

离心淘洗制备不同细胞周期的原发性急性淋巴细胞白血病细胞

The ability to synchronize cells has been central to advancing our understanding of cell cycle regulation. Common techniques employed include serum ...

Flow Cytometry to Estimate Leukemia Stem Cells in Primary Acute Myeloid Leukemia and in Patient-derived-xenografts, at Diagnosis and Follow Up

流式细胞仪估测原发性急性髓系白血病和患者源性移植中白血病干细胞的诊断和随访

Acute myeloid leukemia (AML) is a heterogeneous, and if not treated, fatal disease. It is the most common cause of leukemia-associated mortality in ...

Immunoglobulin Gene Sequence Analysis In Chronic Lymphocytic Leukemia: From Patient Material To Sequence Interpretation

慢性淋巴细胞白血病免疫球蛋白基因序列分析: 从患者材料到序列解释

During B cell maturation, the complex process of immunoglobulin (IG) gene V(D)J recombination coupled with somatic hypermutation (SHM) gives rise to a ...

A Chromatin Immunoprecipitation Assay to Identify Novel NFAT2 Target Genes in Chronic Lymphocytic Leukemia

一种检测慢性淋巴细胞白血病新 nfat2 靶基因的染色质免疫沉淀法

Chronic lymphocytic leukemia (CLL) is characterized by the expansion of malignant B cell clones and represents the most common leukemia in western ...

Assessment of the Metabolic Profile of Primary Leukemia Cells

原发性白血病细胞代谢谱的评价

The metabolic requirement of cancer cells can negatively influence survival and treatment efficacy. Nowadays, pharmaceutical targeting of metabolic ...

Two Flow Cytometric Approaches of NKG2D Ligand Surface Detection to Distinguish Stem Cells from Bulk Subpopulations in Acute Myeloid Leukemia

NKG2D 利甘德表面检测的两种流细胞学方法，以区分干细胞与急性骨髓性白血病中的散装亚群

Within the same patient, absence of NKG2D ligands (NKG2DL) surface expression was shown to distinguish leukemic subpopulations with stem cell properties ...

Tumor Transplantation for Assessing the Dynamics of Tumor-Infiltrating CD8+ T Cells in Mice

Tumor Transplantation for Assessing the Dynamics of Tumor-Infiltrating CD8+ T Cells in Mice

肿瘤移植用于评估小鼠肿瘤浸润CD8 + T细胞的动力学

T cell-mediated immunity plays a crucial role in immune responses against tumors, with cytotoxic T lymphocytes (CTLs) playing the leading role in ...

Subculture and Cryopreservation of Esophageal Adenocarcinoma Organoids: Pros and Cons for Single Cell Digestion

食管腺癌类器官的传代培养和冷冻保存：单细胞消化的利弊

The lack of suitable translational research models reflecting primary disease to explore tumorigenesis and therapeutic strategies is a major obstacle in ...