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Protocol:Liposomal Clodronate-mediated Macrophage Depletion in the Zebrafish Model

作者:   發(fā)布于:2023年08月18日  點(diǎn)擊量:1196

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概要

進(jìn)行體內(nèi)巨噬細(xì)胞特異性耗竭的能力仍然是在廣泛的生理背景下揭示巨噬細(xì)胞功能的有效手段。與小鼠模型相比,斑馬魚(yú)具有卓越的成像能力,因?yàn)樗鼈儚膯渭?xì)胞階段到整個(gè)幼蟲(chóng)發(fā)育過(guò)程中都具有光學(xué)透明度。這些品質(zhì)對(duì)于體內(nèi)細(xì)胞特異性耗竭變得很重要,因此可以通過(guò)顯微鏡實(shí)時(shí)跟蹤和驗(yàn)證目標(biāo)細(xì)胞的消除。有多種方法可以去除斑馬魚(yú)中的巨噬細(xì)胞,包括遺傳(例如 irf8 敲除)、化學(xué)遺傳(例如硝基還原酶/甲硝唑系統(tǒng))和基于毒素的耗竭(例如使用氯膦酸鹽脂質(zhì)體)。在吞噬脂質(zhì)體后使用含氯膦酸鹽的脂質(zhì)體誘導(dǎo)巨噬細(xì)胞凋亡可有效消耗巨噬細(xì)胞以及測(cè)試其吞噬能力。在這里,我們描述了通過(guò)靜脈注射補(bǔ)充有熒光葡聚糖偶聯(lián)物的氯膦酸脂質(zhì)體來(lái)全身耗竭斑馬魚(yú)幼蟲(chóng)巨噬細(xì)胞的詳細(xì)方案。與熒光葡聚糖共注射可以實(shí)時(shí)跟蹤巨噬細(xì)胞耗竭,從驗(yàn)證成功靜脈注射到巨噬細(xì)胞分子攝取及其最終死亡開(kāi)始。為了驗(yàn)證巨噬細(xì)胞的高度耗竭,當(dāng)在早期幼蟲(chóng)階段進(jìn)行氯膦酸鹽注射時(shí),可以通過(guò)快速中性紅色活體染料染色來(lái)確定腦巨噬細(xì)胞(小膠質(zhì)細(xì)胞)消除的水平。

Bio-Protocol-Liposomal Clodronate-mediated Macrophage Depletion in the Zebrafish Model

Experimental workflow for in vivo macrophage-specific depletion by liposomal clodronate in larval zebrafish

背景

巨噬細(xì)胞是先天免疫系統(tǒng)的關(guān)鍵成分,在應(yīng)對(duì)感染、無(wú)菌炎癥和環(huán)境變化方面發(fā)揮著重要作用。將巨噬細(xì)胞的功能與不同生理環(huán)境中相互作用的細(xì)胞類(lèi)型的復(fù)雜組合解耦的最有效方法之一是能夠特異性地消除巨噬細(xì)胞并分析表型后果。小鼠的這種耗竭實(shí)驗(yàn)為巨噬細(xì)胞的作用提供了很多見(jiàn)解(Hua et al., 2018; Rosowski, 2020)。然而,我們對(duì)巨噬細(xì)胞功能的理解仍然不完整,小鼠模型中的細(xì)胞耗竭實(shí)驗(yàn)難以實(shí)時(shí)跟蹤和驗(yàn)證。由于這些原因,斑馬魚(yú)幼蟲(chóng)的光學(xué)透明度和易于操作性通過(guò)對(duì)靶細(xì)胞和整個(gè)完整生物體進(jìn)行實(shí)時(shí)成像,為體內(nèi)高度可追溯和可處理的細(xì)胞消融提供了明顯的優(yōu)勢(shì)。斑馬魚(yú)的基因和免疫系統(tǒng)也與人類(lèi)的基因和免疫系統(tǒng)具有高度的正統(tǒng)性(Yoder et al., 2002; Santoriello et al., 2012; Howe et al., 2013)。此外,斑馬魚(yú)的適應(yīng)性免疫系統(tǒng)直到幼年成年階段才在功能上成熟(Lam et al., 2004),這使得斑馬魚(yú)幼蟲(chóng)成為研究獨(dú)立于適應(yīng)性免疫貢獻(xiàn)的先天免疫系統(tǒng)的絕佳平臺(tái)。

斑馬魚(yú)目前可用的巨噬細(xì)胞耗竭方法包括遺傳和化學(xué)遺傳操作,以及基于毒素的耗竭。巨噬細(xì)胞的發(fā)育需要轉(zhuǎn)錄因子Pu.1(基因名稱(chēng)為spi1b)以及另一種轉(zhuǎn)錄因子Irf8的早期和持續(xù)功能(Li et al., 2011; Shiau et al., 2015; Tenor et al., 2015)。通過(guò)基因敲除或嗎啉諾(MO)反義低聚物敲低PU.1或irf8的破壞,為巨噬細(xì)胞耗竭提供了一種可靠的方法,而前者消融骨髓細(xì)胞,后者對(duì)巨噬細(xì)胞更具特異性,但也會(huì)導(dǎo)致中性粒細(xì)胞數(shù)量的增加(Shiau et al., 2015; Yang et al., 2020)。這些方法不適合時(shí)間控制(Rhodes et al., 2005; Li et al., 2011; Shiau et al., 2015; Rosowski, 2020),而氯膦酸鹽介導(dǎo)的基于局部顯微注射的巨噬細(xì)胞耗竭可以實(shí)現(xiàn)一定程度的空間和時(shí)間指定(Bernut et al., 2014)。

氯膦酸鹽(也稱(chēng)為二氯亞甲基二膦酸鹽)可以被細(xì)胞代謝以阻斷線粒體呼吸,這是由于形成不可水解的ATP類(lèi)似物,然后導(dǎo)致細(xì)胞死亡(細(xì)胞凋亡)(Rosowski, 2020)。一旦注射包封在脂質(zhì)體中,氯膦酸鹽很容易被巨噬細(xì)胞攝入和消除,因?yàn)樗诩?xì)胞內(nèi)積聚(van Rooijen and Hendrikx, 2010)。由于所使用的氯膦酸鹽和脂質(zhì)體磷脂對(duì)其他非吞噬細(xì)胞都沒(méi)有毒性(van Rooijen and Hendrikx, 2010),這種方法允許特異性消耗已經(jīng)存在的吞噬巨噬細(xì)胞。

作為我們方案設(shè)計(jì)的一部分,我們將熒光標(biāo)記的葡聚糖與氯膦酸脂質(zhì)體共同注射,以使我們能夠驗(yàn)證精確和準(zhǔn)確的注射,并跟蹤氯膦酸對(duì)整個(gè)幼蟲(chóng)中巨噬細(xì)胞的影響。為此,在氯膦酸脂質(zhì)體與熒光標(biāo)記的葡聚糖靜脈內(nèi)共注射后,我們目視驗(yàn)證了這些物質(zhì)成功注射到循環(huán)中,并監(jiān)測(cè)了巨噬細(xì)胞對(duì)熒光葡聚糖的攝取及其隨時(shí)間推移的最終死亡。我們?cè)O(shè)計(jì)了該方案,包括注射后48小時(shí),以允許氯膦酸誘導(dǎo)巨噬細(xì)胞凋亡的作用實(shí)現(xiàn),因?yàn)橄惹霸陔u和小鼠中的工作表明氯膦酸的功效可能需要幾天時(shí)間,具體取決于組織(Kameka et al., 2014; Ponzoni et al., 2018)。我們通過(guò)評(píng)估腦駐留巨噬細(xì)胞(小膠質(zhì)細(xì)胞)的剩余數(shù)量,證實(shí)了氯膦酸鹽介導(dǎo)的巨噬細(xì)胞耗竭在注射后48小時(shí)內(nèi)的療效,因?yàn)榭梢酝ㄟ^(guò)中性紅色活體染料染色對(duì)活幼蟲(chóng)中的小膠質(zhì)細(xì)胞進(jìn)行快速分析。我們選擇在幼蟲(chóng)早期階段注射3 dpf(受精后幾天),因?yàn)檫@是在血腦屏障成熟之前(Jeong et al., 2008; O’Brown et al., 2019),當(dāng)時(shí)我們發(fā)現(xiàn)我們注射的物質(zhì)很容易到達(dá)包括大腦在內(nèi)的全身巨噬細(xì)胞。使用氯膦酸鹽生效的 48 小時(shí)窗口,我們能夠在大多數(shù)注射的斑馬魚(yú)幼蟲(chóng)中實(shí)現(xiàn)小膠質(zhì)細(xì)胞的完全消融(Yang et al., 2020)??傮w而言,我們發(fā)現(xiàn)以3dpf的48小時(shí)孵育時(shí)間靜脈顯微注射氯膦酸脂質(zhì)體可有效消除巨噬細(xì)胞。


參考文獻(xiàn)

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原始文獻(xiàn)

1. Yang, L., Rojas, A. M. and Shiau, C. E. (2021). Liposomal Clodronate-mediated Macrophage Depletion in the Zebrafish Model. Bio-protocol 11(6): e3951. DOI: 10.21769/BioProtoc.3951.

2. Yang, L., Jimenez, J. A., Earley, A. M., Hamlin, V., Kwon, V., Dixon, C. T. and Shiau, C. E. (2020). Drainage of inflammatory macromolecules from the brain to periphery targets the liver for macrophage infiltration. Elife 9: e58191.