巨噬细胞清除操作及文献实例分析
巨噬细胞是体内每个器官中唯一都存在的细胞，存在于表皮、角膜和没有血管的关节内部，其体内生物学研究的重要方法之一是巨噬细胞耗竭。清除巨噬细胞，可以全面了解巨噬细胞在病理条件下的功能。目前，巨噬细胞清除的主要方法包括构建巨噬细胞缺失动物模型和使用药物（氯膦酸盐脂质体）去除法，但是，构建巨噬细胞缺失动物模型价格昂贵，周期长，因此，氯膦酸盐脂质体（Clodronate Liposomes）是目前最为成熟、方便、经济的一种巨噬细胞清除工具，可以有效的清除动物体内包括肝脏，脾脏，肺部，血液等多个不同组织和部位中的巨噬细胞，也是目前使用最广泛的一种巨噬细胞清除的方法。
将氯膦酸盐包裹进磷脂体的水相中形成氯膦酸盐脂质体，氯膦酸盐不能自由的通过脂质体磷脂体双分子层。注射到生物体内后，氯膦酸盐脂质体会被巨噬细胞吞噬，在巨噬细胞内的溶酶体磷酸酶的作用下，溶解在脂质体内的氯膦酸盐会被逐步释放出来并在细胞内积累，当达到一定浓度时，巨噬细胞将会受到不可逆损伤，诱发细胞凋亡。死亡细胞内的氯膦酸盐会被释放到细胞外，随尿液排出体外。
不同种类的小鼠给药剂量略有差异，可根据以下表格中的给药剂量摸索。
表1.C57小鼠巨噬细胞清除给药方法
巨噬细胞有很多种亚型，不同的亚型部分标志物不同（见表2），另外，不同组织和器官的巨噬细胞的标志物也会有差异（见表3）。一般在做巨噬细胞清除实验前，应先查找相关文献，找出具体清除部位的巨噬细胞的表面标志物，选择最佳标志物进行流式实验。
表2.不同亚型的巨噬细胞标志物
参考文献：Klopfleisch R. Macrophage reaction against biomaterials in the mouse model - Phenotypes, functions and markers. Acta Biomater. 2016 Oct 1;43:3-13. doi: 10.1016/j.actbio.2016.07.003. Epub 2016 Jul 6. PMID: 27395828.
表3.不同组织中巨噬细胞的标志物
小鼠品系：C57BL/6J小鼠
注射方式：腹腔注射
取样部位：外周血
注射剂量：200 μl /只
抗体选择：F4/80
检测方法：巨噬细胞耗竭模型采用氯膦酸脂质体，腹腔注射200μL每只小鼠，在注射氯钠脂质体后24小时，通过流式细胞术检测外周血巨噬细胞数量。
清除结果：
图1.腹腔注射给药后巨噬细胞减少
参考文献：Xiong X, Chen S, Shen J, et al. Cannabis suppresses antitumor immunity by inhibiting JAK/STAT signaling in T cells through CNR2. Signal Transduct Target Ther. 2022;7(1):99. Published 2022 Apr 6. doi:10.1038/s41392-022-00918-y(IF:18.187)
小鼠品系：雌性C57BL/6J小鼠
注射方式：腹腔注射
取样部位：腹腔灌洗液
注射剂量：第0、3、6天分别给与15 mg/kg巨噬细胞清除剂
抗体选择：CD11b，F4/80双阳性
检测方法：在第0、3、6天将氯膦酸脂质体 (15 mg/kg)或悬浮在200 ml PBS中的空脂质体腹腔注射到6周龄雌性C57BL/6J小鼠体内，以消耗巨噬细胞，并在最后一次脂质体注射后的第3、6天将100万BMDMs悬浮在100 ml PBS中静脉注射重建巨噬细胞，然后通过流式细胞术分析小鼠腹腔灌洗液巨噬细胞的损耗和重构。
清除结果：
图2.注射脂质体后鼠腹腔灌洗液及巨噬细胞重建的流式分析
参考文献：Zhang Z, Chen C, Yang F, et al. Itaconate is a lysosomal inducer that promotes antibacterial innate immunity [published online ahead of print, 2022 May 25. Mol Cell. 2022;S1097-2765(22)00443-9. doi:10.1016/j.molcel.2022.05.009 (IF:17.970)
小鼠品系：C57BL/6J
注射方式：静脉注射
取样部位：血液、乳腺
注射剂量：200 μl /只
抗体选择：F4/80
清除方法：巨噬细胞耗竭模型采用氯膦酸脂质体(0.15-0.2 mL/25 g)，给与200μL每只小鼠，静脉注射于哺乳期小鼠体内，消耗血液和乳腺内巨噬细胞，在注射氯钠脂质体后24、48和72小时，通过流式细胞术检测乳腺巨噬细胞数量。
清除结果：
图3.a）血液学自动分析仪检测血液中单核细胞(血液中巨噬细胞的一种形式)比例的变化；b）流式细胞术检测乳腺巨噬细胞比例变化
参考文献：Cai J, Peng J, Zang X, Feng J, Li R, Ren P, Zheng B, Wang J, Wang J, Yan M, Liu J, Deng R, Wang D. Mammary Leukocyte-Assisted Nanoparticle Transport Enhances Targeted Milk Trace Mineral Delivery. Adv Sci (Weinh). 2022 Sep;9(26):e2200841. doi: 10.1002/advs.202200841. Epub 2022 Jun 30. PMID: 35773238; PMCID: PMC9475556. (IF:17.521)
小鼠品系：C57BL/6
注射方式：腹腔注射
取样部位：外周血
注射剂量：200 μl /只
抗体选择：F4/80
检测方法： 第3天腹腔注射氯磷酸脂质体200 μL，去除巨噬细胞。用流式细胞术检测巨噬细胞缺失效应。
清除结果：
图4.免疫细胞缺失流式分析
参考文献：Yu X, Long Y, Chen B, et al. PD-L1/TLR7 dual-targeting nanobody-drug conjugate mediates potent tumor regression via elevating tumor immunogenicity in a host-expressed PD-L1 bias-dependent way. J Immunother Cancer. 2022;10(10):e004590. doi:10.1136/jitc-2022-004590(IF:12.469)
产品简介
产品组成：1 mL脂质体体悬液（1.5-2μm）中所含的氯膦酸大约为5 mg，所用的磷酸盐缓冲液为10 mM Na2HPO4，10 mM NaH2PO4，140 mM NaCl。
产品列表
|
产品名称 |
货号 |
规格 |
|
Clodronate Liposomes（From Vrije Universiteit Amsterdam）体内巨噬细胞清除剂 |
40337ES08 |
5 mL |
|
40337ES10 |
10 mL |
|
|
40338ES08 |
5 mL |
|
|
40338ES10 |
10 mL |
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