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概要

進(jin)行體內巨(ju)(ju)(ju)(ju)噬(shi)細(xi)(xi)(xi)(xi)胞特異(yi)性耗(hao)竭的能(neng)力仍然是在(zai)(zai)廣泛的生理(li)背(bei)景(jing)下揭示巨(ju)(ju)(ju)(ju)噬(shi)細(xi)(xi)(xi)(xi)胞功能(neng)的有(you)(you)效(xiao)手段。與小鼠(shu)模型(xing)相比，斑(ban)馬(ma)(ma)(ma)魚(yu)(yu)具有(you)(you)卓越的成(cheng)像能(neng)力，因(yin)為(wei)它們(men)從單細(xi)(xi)(xi)(xi)胞階(jie)段到整個(ge)幼(you)蟲發育過(guo)(guo)程中(zhong)(zhong)都具有(you)(you)光學透(tou)明(ming)度。這些品(pin)質(zhi)對(dui)于體內細(xi)(xi)(xi)(xi)胞特異(yi)性耗(hao)竭變得很重要，因(yin)此(ci)可(ke)以通(tong)過(guo)(guo)顯(xian)微鏡實(shi)時跟蹤和(he)驗證目標細(xi)(xi)(xi)(xi)胞的消(xiao)(xiao)除(chu)。有(you)(you)多種方法可(ke)以去除(chu)斑(ban)馬(ma)(ma)(ma)魚(yu)(yu)中(zhong)(zhong)的巨(ju)(ju)(ju)(ju)噬(shi)細(xi)(xi)(xi)(xi)胞，包括遺(yi)傳(chuan)（例如 irf8 敲除(chu)）、化(hua)學遺(yi)傳(chuan)（例如硝(xiao)基(ji)還原(yuan)酶/甲硝(xiao)唑系統）和(he)基(ji)于毒(du)素的耗(hao)竭（例如使(shi)用氯(lv)膦(lin)酸(suan)鹽脂(zhi)質(zhi)體）。在(zai)(zai)吞噬(shi)脂(zhi)質(zhi)體后使(shi)用含(han)氯(lv)膦(lin)酸(suan)鹽的脂(zhi)質(zhi)體誘導巨(ju)(ju)(ju)(ju)噬(shi)細(xi)(xi)(xi)(xi)胞凋(diao)亡可(ke)有(you)(you)效(xiao)消(xiao)(xiao)耗(hao)巨(ju)(ju)(ju)(ju)噬(shi)細(xi)(xi)(xi)(xi)胞以及測試其(qi)吞噬(shi)能(neng)力。在(zai)(zai)這里，我們(men)描述了通(tong)過(guo)(guo)靜脈(mo)注(zhu)(zhu)射(she)補(bu)充有(you)(you)熒光葡(pu)聚糖偶聯(lian)物的氯(lv)膦(lin)酸(suan)脂(zhi)質(zhi)體來全身耗(hao)竭斑(ban)馬(ma)(ma)(ma)魚(yu)(yu)幼(you)蟲巨(ju)(ju)(ju)(ju)噬(shi)細(xi)(xi)(xi)(xi)胞的詳細(xi)(xi)(xi)(xi)方案。與熒光葡(pu)聚糖共注(zhu)(zhu)射(she)可(ke)以實(shi)時跟蹤巨(ju)(ju)(ju)(ju)噬(shi)細(xi)(xi)(xi)(xi)胞耗(hao)竭，從驗證成(cheng)功靜脈(mo)注(zhu)(zhu)射(she)到巨(ju)(ju)(ju)(ju)噬(shi)細(xi)(xi)(xi)(xi)胞分(fen)子攝取及其(qi)最終(zhong)死亡開始。為(wei)了驗證巨(ju)(ju)(ju)(ju)噬(shi)細(xi)(xi)(xi)(xi)胞的高(gao)度耗(hao)竭，當在(zai)(zai)早期幼(you)蟲階(jie)段進(jin)行氯(lv)膦(lin)酸(suan)鹽注(zhu)(zhu)射(she)時，可(ke)以通(tong)過(guo)(guo)快(kuai)速中(zhong)(zhong)性紅色活體染料(liao)染色來確定腦巨(ju)(ju)(ju)(ju)噬(shi)細(xi)(xi)(xi)(xi)胞（小膠質(zhi)細(xi)(xi)(xi)(xi)胞）消(xiao)(xiao)除(chu)的水平。

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

背景(jing)

巨(ju)噬(shi)細(xi)(xi)胞(bao)(bao)是先天免(mian)疫(yi)(yi)(yi)(yi)系(xi)統的(de)(de)(de)(de)關鍵成(cheng)分(fen)，在應對感染、無菌炎癥和(he)(he)(he)環境變化方面發揮著重要作(zuo)用。將巨(ju)噬(shi)細(xi)(xi)胞(bao)(bao)的(de)(de)(de)(de)功(gong)(gong)能(neng)(neng)與(yu)不(bu)同生(sheng)理(li)環境中相(xiang)互作(zuo)用的(de)(de)(de)(de)細(xi)(xi)胞(bao)(bao)類(lei)型(xing)的(de)(de)(de)(de)復(fu)雜組合(he)解(jie)耦的(de)(de)(de)(de)最(zui)有效方法之一是能(neng)(neng)夠(gou)特異(yi)性地消(xiao)除巨(ju)噬(shi)細(xi)(xi)胞(bao)(bao)并(bing)分(fen)析表型(xing)后果。小(xiao)鼠的(de)(de)(de)(de)這(zhe)種(zhong)耗竭實(shi)驗為巨(ju)噬(shi)細(xi)(xi)胞(bao)(bao)的(de)(de)(de)(de)作(zuo)用提供(gong)了很多(duo)見解(jie)（Hua et al., 2018; Rosowski, 2020）。然(ran)而，我們對巨(ju)噬(shi)細(xi)(xi)胞(bao)(bao)功(gong)(gong)能(neng)(neng)的(de)(de)(de)(de)理(li)解(jie)仍然(ran)不(bu)完(wan)整(zheng)，小(xiao)鼠模(mo)型(xing)中的(de)(de)(de)(de)細(xi)(xi)胞(bao)(bao)耗竭實(shi)驗難(nan)以實(shi)時跟蹤和(he)(he)(he)驗證。由于這(zhe)些原因(yin)(yin)，斑馬魚(yu)幼蟲的(de)(de)(de)(de)光(guang)學透明度(du)和(he)(he)(he)易于操作(zuo)性通(tong)過對靶(ba)細(xi)(xi)胞(bao)(bao)和(he)(he)(he)整(zheng)個完(wan)整(zheng)生(sheng)物體進(jin)行實(shi)時成(cheng)像，為體內高度(du)可(ke)追溯(su)和(he)(he)(he)可(ke)處理(li)的(de)(de)(de)(de)細(xi)(xi)胞(bao)(bao)消(xiao)融提供(gong)了明顯的(de)(de)(de)(de)優勢(shi)。斑馬魚(yu)的(de)(de)(de)(de)基因(yin)(yin)和(he)(he)(he)免(mian)疫(yi)(yi)(yi)(yi)系(xi)統也與(yu)人(ren)類(lei)的(de)(de)(de)(de)基因(yin)(yin)和(he)(he)(he)免(mian)疫(yi)(yi)(yi)(yi)系(xi)統具有高度(du)的(de)(de)(de)(de)正統性（Yoder et al., 2002; Santoriello et al., 2012; Howe et al., 2013）。此(ci)外，斑馬魚(yu)的(de)(de)(de)(de)適應性免(mian)疫(yi)(yi)(yi)(yi)系(xi)統直到幼年成(cheng)年階段才在功(gong)(gong)能(neng)(neng)上(shang)成(cheng)熟（Lam et al., 2004），這(zhe)使得斑馬魚(yu)幼蟲成(cheng)為研究獨(du)立于適應性免(mian)疫(yi)(yi)(yi)(yi)貢獻的(de)(de)(de)(de)先天免(mian)疫(yi)(yi)(yi)(yi)系(xi)統的(de)(de)(de)(de)絕佳(jia)平臺。

斑馬魚(yu)目前可(ke)(ke)用的(de)(de)(de)巨噬細胞耗(hao)竭方(fang)法(fa)包(bao)括遺傳和化(hua)學遺傳操作，以(yi)(yi)及基(ji)(ji)于毒素的(de)(de)(de)耗(hao)竭。巨噬細胞的(de)(de)(de)發(fa)育(yu)需要轉錄因(yin)子Pu.1（基(ji)(ji)因(yin)名稱為spi1b）以(yi)(yi)及另一(yi)種轉錄因(yin)子Irf8的(de)(de)(de)早期(qi)和持(chi)續(xu)功能（Li et al., 2011; Shiau et al., 2015; Tenor et al., 2015）。通過(guo)基(ji)(ji)因(yin)敲除或嗎啉諾（MO）反義低(di)聚物敲低(di)PU.1或irf8的(de)(de)(de)破壞(huai)，為巨噬細胞耗(hao)竭提供(gong)了一(yi)種可(ke)(ke)靠的(de)(de)(de)方(fang)法(fa)，而前者消融骨髓細胞，后者對巨噬細胞更具(ju)特異性(xing)，但也(ye)會導(dao)致中性(xing)粒細胞數量(liang)的(de)(de)(de)增加(jia)（Shiau et al., 2015; Yang et al., 2020）。這些方(fang)法(fa)不(bu)適合時(shi)間(jian)控(kong)制（Rhodes et al., 2005; Li et al., 2011; Shiau et al., 2015; Rosowski, 2020），而氯膦酸鹽介導(dao)的(de)(de)(de)基(ji)(ji)于局部(bu)顯微注射的(de)(de)(de)巨噬細胞耗(hao)竭可(ke)(ke)以(yi)(yi)實現一(yi)定程度的(de)(de)(de)空間(jian)和時(shi)間(jian)指定（Bernut et al., 2014）。

氯(lv)膦(lin)(lin)(lin)酸鹽(yan)（也稱(cheng)為二氯(lv)亞甲基(ji)二膦(lin)(lin)(lin)酸鹽(yan)）可以被細(xi)胞(bao)(bao)(bao)代謝以阻斷線粒體呼吸，這(zhe)是由于形成不可水解的(de)ATP類似物，然后導致細(xi)胞(bao)(bao)(bao)死亡（細(xi)胞(bao)(bao)(bao)凋亡）（Rosowski, 2020）。一旦注射包封(feng)在脂質體中，氯(lv)膦(lin)(lin)(lin)酸鹽(yan)很容易(yi)被巨噬(shi)細(xi)胞(bao)(bao)(bao)攝入和(he)消除，因(yin)為它在細(xi)胞(bao)(bao)(bao)內(nei)積(ji)聚（van Rooijen and Hendrikx, 2010）。由于所使用的(de)氯(lv)膦(lin)(lin)(lin)酸鹽(yan)和(he)脂質體磷脂對其他非(fei)吞(tun)噬(shi)細(xi)胞(bao)(bao)(bao)都沒有毒性（van Rooijen and Hendrikx, 2010），這(zhe)種(zhong)方法(fa)允許特異性消耗(hao)已(yi)經存(cun)在的(de)吞(tun)噬(shi)巨噬(shi)細(xi)胞(bao)(bao)(bao)。

作(zuo)為我(wo)(wo)(wo)們方案(an)設計的(de)(de)(de)(de)一(yi)部分，我(wo)(wo)(wo)們將熒(ying)光(guang)(guang)標(biao)記(ji)的(de)(de)(de)(de)葡聚糖與氯膦(lin)酸(suan)(suan)(suan)脂(zhi)質(zhi)(zhi)體共同注(zhu)射，以使我(wo)(wo)(wo)們能夠驗證精確(que)和(he)準確(que)的(de)(de)(de)(de)注(zhu)射，并(bing)跟蹤氯膦(lin)酸(suan)(suan)(suan)對整個幼(you)蟲(chong)中(zhong)巨(ju)(ju)噬(shi)(shi)細(xi)(xi)(xi)(xi)胞(bao)(bao)(bao)的(de)(de)(de)(de)影響。為此，在氯膦(lin)酸(suan)(suan)(suan)脂(zhi)質(zhi)(zhi)體與熒(ying)光(guang)(guang)標(biao)記(ji)的(de)(de)(de)(de)葡聚糖靜(jing)脈內(nei)共注(zhu)射后(hou)，我(wo)(wo)(wo)們目視驗證了這(zhe)些物質(zhi)(zhi)成功注(zhu)射到循(xun)環中(zhong)，并(bing)監測(ce)了巨(ju)(ju)噬(shi)(shi)細(xi)(xi)(xi)(xi)胞(bao)(bao)(bao)對熒(ying)光(guang)(guang)葡聚糖的(de)(de)(de)(de)攝取(qu)及(ji)其隨時(shi)(shi)(shi)間推移的(de)(de)(de)(de)最(zui)終(zhong)死亡。我(wo)(wo)(wo)們設計了該方案(an)，包括注(zhu)射后(hou)48小(xiao)時(shi)(shi)(shi)，以允許(xu)氯膦(lin)酸(suan)(suan)(suan)誘導(dao)巨(ju)(ju)噬(shi)(shi)細(xi)(xi)(xi)(xi)胞(bao)(bao)(bao)凋亡的(de)(de)(de)(de)作(zuo)用實(shi)現，因(yin)為先前在雞和(he)小(xiao)鼠中(zhong)的(de)(de)(de)(de)工作(zuo)表明氯膦(lin)酸(suan)(suan)(suan)的(de)(de)(de)(de)功效(xiao)(xiao)可能需要幾(ji)天時(shi)(shi)(shi)間，具(ju)體取(qu)決于組(zu)織(zhi)（Kameka et al., 2014; Ponzoni et al., 2018）。我(wo)(wo)(wo)們通(tong)過(guo)評估腦(nao)駐(zhu)留(liu)巨(ju)(ju)噬(shi)(shi)細(xi)(xi)(xi)(xi)胞(bao)(bao)(bao)（小(xiao)膠(jiao)質(zhi)(zhi)細(xi)(xi)(xi)(xi)胞(bao)(bao)(bao)）的(de)(de)(de)(de)剩余數量，證實(shi)了氯膦(lin)酸(suan)(suan)(suan)鹽(yan)介導(dao)的(de)(de)(de)(de)巨(ju)(ju)噬(shi)(shi)細(xi)(xi)(xi)(xi)胞(bao)(bao)(bao)耗竭(jie)在注(zhu)射后(hou)48小(xiao)時(shi)(shi)(shi)內(nei)的(de)(de)(de)(de)療效(xiao)(xiao)，因(yin)為可以通(tong)過(guo)中(zhong)性紅色活體染(ran)料染(ran)色對活幼(you)蟲(chong)中(zhong)的(de)(de)(de)(de)小(xiao)膠(jiao)質(zhi)(zhi)細(xi)(xi)(xi)(xi)胞(bao)(bao)(bao)進行(xing)快速分析。我(wo)(wo)(wo)們選擇(ze)在幼(you)蟲(chong)早期階段注(zhu)射3 dpf（受(shou)精后(hou)幾(ji)天），因(yin)為這(zhe)是在血(xue)腦(nao)屏障成熟之前（Jeong et al., 2008; O’Brown et al., 2019），當時(shi)(shi)(shi)我(wo)(wo)(wo)們發(fa)(fa)現我(wo)(wo)(wo)們注(zhu)射的(de)(de)(de)(de)物質(zhi)(zhi)很(hen)容(rong)易到達(da)包括大腦(nao)在內(nei)的(de)(de)(de)(de)全身巨(ju)(ju)噬(shi)(shi)細(xi)(xi)(xi)(xi)胞(bao)(bao)(bao)。使用氯膦(lin)酸(suan)(suan)(suan)鹽(yan)生效(xiao)(xiao)的(de)(de)(de)(de) 48 小(xiao)時(shi)(shi)(shi)窗(chuang)口，我(wo)(wo)(wo)們能夠在大多數注(zhu)射的(de)(de)(de)(de)斑馬(ma)魚幼(you)蟲(chong)中(zhong)實(shi)現小(xiao)膠(jiao)質(zhi)(zhi)細(xi)(xi)(xi)(xi)胞(bao)(bao)(bao)的(de)(de)(de)(de)完全消(xiao)(xiao)融（Yang et al., 2020）。總體而言，我(wo)(wo)(wo)們發(fa)(fa)現以3dpf的(de)(de)(de)(de)48小(xiao)時(shi)(shi)(shi)孵育時(shi)(shi)(shi)間靜(jing)脈顯微注(zhu)射氯膦(lin)酸(suan)(suan)(suan)脂(zhi)質(zhi)(zhi)體可有(you)效(xiao)(xiao)消(xiao)(xiao)除巨(ju)(ju)噬(shi)(shi)細(xi)(xi)(xi)(xi)胞(bao)(bao)(bao)。

參考文獻

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原始文獻

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.