胚胎來(lái)源巨噬細(xì)胞的起源及其在肝臟中功能的研究進(jìn)展

王咪咪， 王艷紅

復(fù)旦大學(xué)附屬中山醫(yī)院肝內(nèi)科，上海 200032

巨噬細(xì)胞是機(jī)體免疫系統(tǒng)的重要組成部分，其表型和功能具有高度異質(zhì)性[1-2]。組織定居巨噬細(xì)胞長(zhǎng)期存在于機(jī)體各組織器官，如肝臟中的枯否細(xì)胞、肺組織中的肺泡巨噬細(xì)胞、表皮中的朗格漢斯細(xì)胞、骨組織中的破骨細(xì)胞等，參與機(jī)體的免疫監(jiān)視、免疫防御、免疫自穩(wěn)，具有組織特異性。早期研究認(rèn)為，組織定居巨噬細(xì)胞來(lái)源于成體骨髓單核細(xì)胞；而最新研究發(fā)現(xiàn)，大部分組織的定居巨噬細(xì)胞是在胚胎時(shí)期由胚胎前體細(xì)胞分化而來(lái)的，這種胚胎來(lái)源巨噬細(xì)胞(EDMs)可以通過(guò)自我增殖維持?jǐn)?shù)量和功能穩(wěn)定[3]。這一發(fā)現(xiàn)給巨噬細(xì)胞領(lǐng)域提供了新的研究方向。因此，本文將對(duì)肝臟中胚胎來(lái)源的組織定居巨噬細(xì)胞的起源和功能進(jìn)行綜述，并由此思考和討論目前巨噬細(xì)胞靶向治療的不足和發(fā)展方向。

1 組織定居巨噬細(xì)胞的起源

組織定居巨噬細(xì)胞一直被認(rèn)為是由成體骨髓單核細(xì)胞分化而來(lái)的，即骨髓中的造血干細(xì)胞分化為單核細(xì)胞，后者通過(guò)血液循環(huán)按照不同的周期進(jìn)入不同的組織器官，分化為組織定居巨噬細(xì)胞。隨著細(xì)胞發(fā)育制圖技術(shù)[4]等新技術(shù)的研究應(yīng)用，對(duì)組織定居巨噬細(xì)胞的起源有了新的認(rèn)識(shí)。一般認(rèn)為，在出生之前，胚胎前體細(xì)胞就已分化形成組織定居巨噬細(xì)胞，稱(chēng)為EDMs。這種細(xì)胞的發(fā)育途徑主要分為3個(gè)連續(xù)的造血波[5]，分別命名為原始造血、暫時(shí)決定性造血和決定性造血，分別起源于中胚層背側(cè)板胚外卵黃囊中的血島、卵黃囊生血內(nèi)皮細(xì)胞和胚胎內(nèi)生血內(nèi)皮細(xì)胞在主動(dòng)脈旁臟壁層區(qū)域產(chǎn)生的未成熟的造血干細(xì)胞，分別產(chǎn)生卵黃囊巨噬細(xì)胞、胎肝單核細(xì)胞來(lái)源的巨噬細(xì)胞和胎肝單核細(xì)胞。它們都可在機(jī)體出生前遷移至器官組織中直接增殖或分化為組織定居巨噬細(xì)胞。

2 EDMs的自我更新與穩(wěn)態(tài)維持

“自我更新”是廣泛應(yīng)用于干細(xì)胞的概念，即在長(zhǎng)期不斷的增殖過(guò)程中，子細(xì)胞仍能保留母細(xì)胞特性[3]。靜息狀態(tài)下，大部分組織器官(腸、皮膚、心臟、胰腺等)中EDMs無(wú)法單獨(dú)通過(guò)局部自我增殖以維持?jǐn)?shù)量，需要骨髓造血干細(xì)胞的分化補(bǔ)充[5]。因此，大部分組織定居巨噬細(xì)胞群由EDMs和骨髓單核細(xì)胞來(lái)源的巨噬細(xì)胞共同構(gòu)成。而肝組織定居巨噬細(xì)胞的來(lái)源中，EDMs占絕大多數(shù)，并通過(guò)局部自我更新和增殖維持?jǐn)?shù)量，而不依賴(lài)于骨髓單核細(xì)胞的補(bǔ)充。有研究[6]報(bào)道，肝EDMs的自我更新功能在肝臟微環(huán)境穩(wěn)態(tài)下可以長(zhǎng)期保持，不隨年齡增長(zhǎng)而逐漸消失。但當(dāng)其離開(kāi)肝臟微環(huán)境，就會(huì)喪失自我更新功能[7]。

當(dāng)機(jī)體遇到刺激后，EDMs會(huì)發(fā)生不同程度的壞死，嚴(yán)重時(shí)候數(shù)量甚至?xí)抵翞?(消失期)[8-9]，再通過(guò)局部自我增殖恢復(fù)數(shù)量。由于刺激因素的種類(lèi)、強(qiáng)度、時(shí)間的不同，其恢復(fù)水平和能力會(huì)有所變化。研究顯示，非基因毒性刺激(如白喉毒素、氯磷酸二鈉脂質(zhì)體或寄生蟲(chóng)感染)下，EDMs可局部自我增殖，并在IL-4的作用下極化為M2型，此時(shí)不需要骨髓單核細(xì)胞的分化補(bǔ)充[10-11]；而基因毒性(如致死性放射、骨髓移植[11])，李斯特菌、沙門(mén)桿菌等胞內(nèi)菌感染，病毒感染等刺激下，壞死的巨噬細(xì)胞可通過(guò)分泌CCL2等趨化因子，誘導(dǎo)單核細(xì)胞浸潤(rùn)肝組織，從而代替EDMs，形成新的組織定居巨噬細(xì)胞[8-9,12-13]，此后，肝臟定居巨噬細(xì)胞池的構(gòu)成比例發(fā)生變化，維持穩(wěn)態(tài)的能力下降。另外，動(dòng)物實(shí)驗(yàn)[14]表明，如果調(diào)整刺激強(qiáng)度(細(xì)菌載量或放射劑量)，肝臟內(nèi)會(huì)出現(xiàn)EDMs與骨髓來(lái)源的巨噬細(xì)胞嵌合的狀態(tài)。以上3種情況顯示，一旦穩(wěn)態(tài)被打破，巨噬細(xì)胞池的構(gòu)成將根據(jù)微環(huán)境的不同而發(fā)生變化，進(jìn)而導(dǎo)致不同表現(xiàn)的炎癥反應(yīng)。

3 EDMs介導(dǎo)炎癥反應(yīng)

在肝臟中，EDMs位于肝血竇內(nèi)，黏附于肝血竇內(nèi)皮細(xì)胞上，直接暴露在血液中，是對(duì)損傷或刺激產(chǎn)生反應(yīng)的一線(xiàn)細(xì)胞[15]，較骨髓單核系統(tǒng)來(lái)源的巨噬細(xì)胞能夠更快速更有效地吞噬衰老細(xì)胞、細(xì)菌、病毒等。EDMs通過(guò)其強(qiáng)大的吞噬功能，控制有害物質(zhì)播散[16-18]，減少過(guò)度炎癥反應(yīng)對(duì)肝臟造成的損傷[16]。

病毒、細(xì)菌和壞死的肝細(xì)胞均能釋放損傷相關(guān)分子模式(DAMPs)、病原體相關(guān)分子模式(PAMPs)，后者通過(guò)與清道夫受體(SPs)、toll樣受體(TLRs)、髓細(xì)胞觸發(fā)受體-1(TREM-1)[19]等模式受體結(jié)合激活EDMs，使其極化為M1型巨噬細(xì)胞，并分泌白細(xì)胞介素1(IL-1)、IL-6、腫瘤壞死因子-α(TNF-α)和粒細(xì)胞-巨噬細(xì)胞集落刺激因子(GM-CSF)等促炎因子[20]。此外，一些胞內(nèi)菌(如李斯特菌、沙門(mén)桿菌)、X線(xiàn)照射或N-乙酰對(duì)氨基苯酚[21]等會(huì)導(dǎo)致EDMs大量壞死進(jìn)而激發(fā)單核細(xì)胞、中性粒細(xì)胞的浸潤(rùn)[8,22]，介導(dǎo)炎癥反應(yīng)的發(fā)生。

隨著炎癥反應(yīng)的進(jìn)展，EDMs會(huì)介導(dǎo)抗炎癥反應(yīng)。EDMs可通過(guò)分泌TNF，促進(jìn)中性粒細(xì)胞的凋亡，進(jìn)而吞噬凋亡的中性粒細(xì)胞[23]。對(duì)凋亡中性粒細(xì)胞的吞噬又可促進(jìn)EDMs產(chǎn)生脂質(zhì)介質(zhì)如脂氧素[24]等，抑制中性粒細(xì)胞的進(jìn)一步浸潤(rùn)，從而控制過(guò)度炎癥反應(yīng)。此外，壞死的EDMs可通過(guò)產(chǎn)生IL-1β誘導(dǎo)肝細(xì)胞分泌IL-33，進(jìn)而刺激嗜酸性粒細(xì)胞分泌IL-4；而IL-4能誘導(dǎo)單核來(lái)源的巨噬細(xì)胞從M1型轉(zhuǎn)化為M2型[8]，同時(shí)可作用于尚存的EDMs，使其局部增殖并分化為M2型巨噬細(xì)胞，后者通過(guò)分泌IL-10，活化精氨酸酶，進(jìn)而刺激單核細(xì)胞來(lái)源的M1型巨噬細(xì)胞的凋亡[25]。M2型巨噬細(xì)胞能通過(guò)分泌大量抗炎因子，如TGF-β、IL-10等，削弱炎癥反應(yīng)，介導(dǎo)免疫耐受[26]。

總之，及時(shí)、適度的炎癥有利于清除病原體、凋亡壞死的細(xì)胞以及細(xì)胞碎片等，而過(guò)度的炎癥反應(yīng)或者持續(xù)的慢性炎癥則是惡性病變的重要危險(xiǎn)因素[27-29]。EDMs在平衡炎癥反應(yīng)和免疫恢復(fù)中發(fā)揮舉足輕重的作用，能夠有效避免病變的發(fā)生。

4 EDMs影響HCC和循環(huán)腫瘤細(xì)胞在肝臟中的生長(zhǎng)

炎癥與腫瘤息息相關(guān)，巨噬細(xì)胞是炎癥相關(guān)的重要細(xì)胞[30]。其中，位于腫瘤組織內(nèi)或腫瘤周?chē)M織的腫瘤相關(guān)性巨噬細(xì)胞(TAMs)[31-34]是一類(lèi)分化終末期的巨噬細(xì)胞[35]。TAMs顯示與M2型相似的分子功能譜[36-37]，在HCC的發(fā)展和轉(zhuǎn)移過(guò)程中起至關(guān)重要的作用[38-40]。有文獻(xiàn)[41-42]認(rèn)為，TAMs是由骨髓單核細(xì)胞分化并在腫瘤微環(huán)境內(nèi)極化為M2型的髓系巨噬細(xì)胞。關(guān)于腫瘤組織中EDMs的功能的相關(guān)報(bào)道不多。有實(shí)驗(yàn)[43]顯示，HCC微環(huán)境中，EDMs的抗原遞呈功能下降、CD86和主要組織相容性復(fù)合體Ⅱ分子(MHC-Ⅱ)表達(dá)下降、細(xì)胞程序死亡配體1(PD-L1)表達(dá)上升。腫瘤中髓系來(lái)源的抑制細(xì)胞(MDSCs)作用于EDMs，抑制其分泌CCL2和IL-18，而促進(jìn)其分泌IL-10和IL-1β，并使其表面正性和負(fù)性共刺激分子失平衡，從而促進(jìn)HCC的發(fā)展。

雖然EDMs在腫瘤發(fā)展后期呈現(xiàn)促進(jìn)腫瘤生長(zhǎng)和轉(zhuǎn)移的作用。但作為一線(xiàn)免疫細(xì)胞又具有抵抗循環(huán)腫瘤細(xì)胞進(jìn)入肝臟的作用[44-48]。其機(jī)制主要有：直接吞噬作用[44-50]，降低腫瘤細(xì)胞對(duì)肝竇的黏附[45]；通過(guò)釋放一氧化氮和活性氧來(lái)上調(diào)死亡因子配體(FASL)，誘導(dǎo)腫瘤細(xì)胞的凋亡[51]；通過(guò)分泌細(xì)胞因子如IL-1、IL-6、IL-8、TNF-α、γ-干擾素(IFN-γ)，或趨化因子MIP-2、IP-10、KC/GRO、MIP-1α、MCP-1等，激活自然殺傷細(xì)胞(NK)[52]和中性粒細(xì)胞[51]，進(jìn)而殺死循環(huán)腫瘤細(xì)胞。

然而，轉(zhuǎn)移灶或預(yù)轉(zhuǎn)移龕的微環(huán)境可以抑制EDMs吞噬殺傷循環(huán)腫瘤細(xì)胞的功能。研究[53-54]報(bào)道，在結(jié)直腸腫瘤肝轉(zhuǎn)移中，當(dāng)骨髓髓系細(xì)胞被募集到轉(zhuǎn)移灶時(shí)，此時(shí)預(yù)轉(zhuǎn)移龕、腫瘤微環(huán)境已經(jīng)開(kāi)始建立，大部分單核細(xì)胞不能分化為巨噬細(xì)胞，滯留為MDSCs，后者通過(guò)上調(diào)EDMs表面的負(fù)性T細(xì)胞共刺激分子PD-L1[53-54]，抑制其吞噬效能[46]。

5 EDMs靶向治療的挑戰(zhàn)

目前靶向巨噬細(xì)胞的多種抗腫瘤藥物[55-57]已被逐漸研發(fā)，有些已經(jīng)進(jìn)入臨床試驗(yàn)階段，表現(xiàn)出良好的臨床效果。這些藥物的作用靶點(diǎn)及機(jī)制有減少單核細(xì)胞浸潤(rùn)、抑制單核來(lái)源巨噬細(xì)胞的增殖、控制單核細(xì)胞的分化成熟、消融巨噬細(xì)胞、增強(qiáng)巨噬細(xì)胞吞噬功能、調(diào)控巨噬細(xì)胞的極化方向、對(duì)促進(jìn)腫瘤發(fā)展的巨噬細(xì)胞進(jìn)行再教育。

上述策略大多針對(duì)單核細(xì)胞及其來(lái)源的巨噬細(xì)胞，不適用于EDMs。EDMs表面不表達(dá)CXCR3和CCR2[58]，阻斷CCL2-CCR2并不影響EDMs的局部增殖。兩種來(lái)源的巨噬細(xì)胞有相互補(bǔ)充的現(xiàn)象。當(dāng)阻斷CCL2/CCR2[59]或CSF1/CSF1R信號(hào)通路后，循環(huán)系統(tǒng)無(wú)法及時(shí)供給足夠的巨噬細(xì)胞，肝臟定居的EDMs可通過(guò)自我增殖補(bǔ)充巨噬細(xì)胞池，從而構(gòu)造免疫抑制的腫瘤微環(huán)境，削弱這條機(jī)制靶向的抗腫瘤作用。因此，對(duì)兩種來(lái)源的巨噬細(xì)胞同時(shí)進(jìn)行消融、極化和再教育將是巨噬細(xì)胞靶向藥的研究方向。

此外，多種治療性單克隆抗體通過(guò)抗體依賴(lài)細(xì)胞介導(dǎo)的細(xì)胞毒作用(ADCC)介導(dǎo)巨噬細(xì)胞吞噬，可以有效地殺傷腫瘤細(xì)胞[60-61]。提高巨噬細(xì)胞吞噬功能的抗腫瘤藥物也在動(dòng)物研究中取得了顯著效果，如抗CD47抗體[62-64]。

然而，這些研究忽略了巨噬細(xì)胞的來(lái)源，而不同來(lái)源的巨噬細(xì)胞的吞噬功能不同。研究[48]指出，EDMs具有獨(dú)特的Dectin-2依賴(lài)的吞噬活性。淋巴細(xì)胞脈絡(luò)叢腦膜炎病毒(LCMV)感染的動(dòng)物模型的研究[65]結(jié)果顯示，EDMs的吞噬水平保持穩(wěn)態(tài)，而單核細(xì)胞的吞噬功能則顯著受損。EDMs能更有效地吞噬乙酰化的低密度脂蛋白，而對(duì)細(xì)菌的吞噬能力則不如單核細(xì)胞來(lái)源巨噬細(xì)胞[14]。最新研究[66]發(fā)現(xiàn)，免疫檢查點(diǎn)抑制劑通過(guò)拮抗TAMs表面的PD-1，加強(qiáng)其對(duì)腫瘤細(xì)胞的吞噬，而這些TAMs經(jīng)實(shí)驗(yàn)證實(shí)屬于單核細(xì)胞來(lái)源，而非胚胎來(lái)源。因而，對(duì)不同來(lái)源巨噬細(xì)胞吞噬功能的深入探索可為抗體治療和免疫治療提供更廣闊的視野。

6 小 結(jié)

肝臟定居巨噬細(xì)胞占非實(shí)質(zhì)細(xì)胞的35%，占全身組織定居巨噬細(xì)胞的80%～90%[23]。因此，巨噬細(xì)胞是肝臟維持穩(wěn)態(tài)、參與炎癥反應(yīng)、調(diào)控生理和病理變化的重要成員。近年來(lái)，對(duì)于肝臟EDMs的起源、維持穩(wěn)態(tài)以及介導(dǎo)炎癥反應(yīng)等生理或病理功能的研究有了一定的進(jìn)展，但對(duì)于其在HCC微環(huán)境中的表型和功能仍有待進(jìn)一步的探索。闡明EDMs在各種不同的生理或病理狀態(tài)下的反應(yīng)和功能，不僅可以為靶向巨噬細(xì)胞治療提供新的思路，也有利于臨床對(duì)肝臟相關(guān)疾病病因認(rèn)識(shí)的提高和治療方法的改進(jìn)。

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