鄭志磊，楊爽

? 綜述 ?

胞外誘捕網(wǎng)與動(dòng)脈粥樣硬化的研究

鄭志磊1，楊爽1

動(dòng)脈粥樣硬化（AS）是危害人類健康的一種常見疾病，目前已成為人類健康的第一殺手。AS的特征是動(dòng)脈內(nèi)膜斑塊形成，斑塊內(nèi)均存在炎性細(xì)胞浸潤(rùn)和脂質(zhì)沉積[1]。近年來(lái)，有研究顯示細(xì)胞外游離的DNA-蛋白復(fù)合物與動(dòng)脈粥樣硬化的發(fā)生及發(fā)展有關(guān)[2]，且可能增加斑塊負(fù)荷[3]，而這種DNA-蛋白復(fù)合物即為胞外誘捕網(wǎng)（ETs）。自該項(xiàng)研究結(jié)果提出以來(lái)，ETs成為了近年來(lái)與動(dòng)脈粥樣硬化研究高度相關(guān)的概念。本文就胞外誘捕網(wǎng)的研究及其與動(dòng)脈粥樣硬化的相關(guān)研究進(jìn)展作一綜述。

1 胞外誘捕網(wǎng)定義

在2004年，Zychlinsky and coauthors發(fā)現(xiàn)中性粒細(xì)胞可以在細(xì)胞外通過一種DNA-蛋白質(zhì)復(fù)合物殺死病原體，這種DNA-蛋白質(zhì)復(fù)合物被Science命名為中性粒細(xì)胞胞外誘捕網(wǎng)（neutrophil extracellular traps，NETs）[4]。NETs是中性粒細(xì)胞在被刺激因素激活后釋放其核內(nèi)的DNA，并與胞漿蛋白結(jié)合釋放至胞外形成的網(wǎng)狀結(jié)構(gòu)，是細(xì)胞經(jīng)刺激后進(jìn)行的一種新型的程序性死亡[5]，這種過程被Steinberg等命名為NETosis[6]，因這種DNA-蛋白質(zhì)被釋放到細(xì)胞外的機(jī)制，不僅只發(fā)生在中性粒細(xì)胞，所以NETosis更確切的說應(yīng)為ETosis。ETs的形成過程是一種新型程序性死亡，既不同于凋亡也不同于壞死，被稱為“ETosis”[7]。ETosis形成的細(xì)胞通常會(huì)出現(xiàn)核染色體解聚，繼而核膜溶解，染色質(zhì)和細(xì)胞質(zhì)中的蛋白酶等混合后以擠壓的方式釋放到細(xì)胞外，最終形成ETs[8]。

2 胞外誘捕網(wǎng)家族

目前發(fā)現(xiàn)能夠形成胞外誘捕網(wǎng)的細(xì)胞有：中性粒細(xì)胞、巨噬細(xì)胞、嗜酸性粒細(xì)胞、嗜堿性粒細(xì)胞、肥大細(xì)胞，單核細(xì)胞其釋放的ETs分別為：NETs[4]，巨噬細(xì)胞胞外誘捕網(wǎng)（METs）[9]，嗜酸性粒細(xì)胞胞外誘捕網(wǎng)（EETs）[10]， 嗜堿性粒細(xì)胞胞外誘捕網(wǎng)（BETs）[11]，肥大細(xì)胞胞外誘捕網(wǎng)(MCETs）[12]，單核細(xì)胞胞外誘捕網(wǎng)[13]。

3 NETs的形態(tài)

NETs有一個(gè)獨(dú)特的超微結(jié)構(gòu)，由直徑為15～17 nm的平滑絲狀結(jié)構(gòu)組成[4]，這種絲狀結(jié)構(gòu)是由修飾過的核小體堆疊而成[14]，而NETs的這種骨架結(jié)構(gòu)上布滿直徑為50 nm的顆粒蛋白和其它細(xì)胞成分[4]，令人驚訝的是，NETs不僅有細(xì)長(zhǎng)的薄絲，也可變成比初始細(xì)胞占據(jù)面積大10～15倍的云霧狀或蜘蛛網(wǎng)狀的結(jié)構(gòu)。提示我們NETs形成時(shí)空間大小的差異可影響NETs的具體形態(tài)[15]。

4 NETs的形成機(jī)制

4.1 NETs誘導(dǎo)物 很多病原性生物均可誘導(dǎo)NETosis的發(fā)生（如細(xì)菌、真菌、原生動(dòng)物和病毒[5]），NETosis的發(fā)生可能由這些病原性生物直接刺激誘導(dǎo)，也可能與這些生物細(xì)胞成分或細(xì)胞分泌的某些成分有關(guān)（如某些微生物成分、脂多糖（LPS）[16,17]，來(lái)自鏈球菌的M1蛋白[18]，亞馬遜利什曼原蟲的脂磷酸聚糖[19]等）。此外某些生理性或免疫性自身產(chǎn)物亦可誘導(dǎo)NETosis的發(fā)生（如TLR4激活的血小板[20]、ROS系統(tǒng)過氧化氫等[5]、抗體[21]和抗原抗體復(fù)合物[22,23]等）。

4.2 NETs形成的分子機(jī)制 NETosis是一種依賴不同刺激物刺激細(xì)胞發(fā)生不同形態(tài)變化，最終導(dǎo)致細(xì)胞死亡的復(fù)雜過程。刺激物不同，NETs的成分和進(jìn)展也有所不同。Neeli等提出MAC-1整合蛋白可能參與中性粒細(xì)胞細(xì)胞核及細(xì)胞膜崩解形成NETs的過程，當(dāng)Mac 1整合素受體被激活，細(xì)胞附著于基底意味著NETosis發(fā)生發(fā)展的開始[24]。然而，在中性粒細(xì)胞接受刺激后啟動(dòng)何種機(jī)制選擇吞噬或NETosis的精確識(shí)別，還有待于進(jìn)一步的研究發(fā)現(xiàn)。

對(duì)ROS產(chǎn)物的依賴性是NETosis發(fā)生的特征之一，其基本步驟已大致明確[25,26]。在激活的過程中，中性粒細(xì)胞通過NADPH氧化酶產(chǎn)生了大量的ROS，F(xiàn)uchs已經(jīng)證實(shí)ROS也是NETs產(chǎn)生的啟動(dòng)者[5]。例如，來(lái)自CGD患者的中性粒細(xì)胞不能產(chǎn)生NETs，因?yàn)镃GD的患者NADPH氧化酶的亞基發(fā)生突變進(jìn)而影響了酶的活性。而且，CGD患者的中性粒細(xì)胞在經(jīng)過H2O2的處理后恢復(fù)了形成NETs的能力[27]。在刺激之后，中性粒細(xì)胞通過常染色體與異染色體的混合完成了染色體的解旋，這個(gè)過程是由儲(chǔ)存在噬天清顆粒中的酶介導(dǎo)的，主要包括中性粒細(xì)胞彈性蛋白酶以及髓過氧化物酶等，它們通過一種還未明確的機(jī)制重新定位到細(xì)胞核上。首先，彈性蛋白酶降解了組蛋白H1和核心組蛋白之間的連接，導(dǎo)致染色體的解旋，解旋的過程可以被髓過氧化物酶強(qiáng)化（MPO），但不依賴后者酶的活性[28,29]。而且，在NETs形成的過程中，組蛋白H3中的精氨酸殘基被瓜氨酸化[30-32]。這組蛋白的瓜氨酸化可以被定位在中性粒細(xì)胞核酸上的PAD4促進(jìn)（peptidylarginine deiminase 4， PAD4）。PAD4基因敲除小鼠的中性粒細(xì)胞失去了釋放NET的能力并且組蛋白的瓜氨酸化也沒被觀察到[33]，隨后，這核膜被破壞，細(xì)胞內(nèi)的染色體擴(kuò)張并且與顆粒抗菌因子混合，最終，細(xì)胞膜破壞釋放NETs[15]。上面提到的這些過程都提示這是一種新型的細(xì)胞死亡過程，但是NETs也能被活的中性粒細(xì)胞通過非氧化依賴的途徑在幾分鐘內(nèi)產(chǎn)生，就像在S.aureus infection中展示的一樣[34,35]，考慮到其它細(xì)胞（如肥大細(xì)胞、嗜堿性粒細(xì)胞、巨噬細(xì)胞）也能產(chǎn)生胞外誘捕網(wǎng)ETs[36]這種新的防御機(jī)制，目前還沒有被研究明白統(tǒng)稱為NETosis。

5 NETs與動(dòng)脈粥樣硬化

在動(dòng)脈粥樣硬化中NETs的成分易被誤認(rèn)為是自身細(xì)胞成分，NETs被發(fā)現(xiàn)存在于人和小鼠的動(dòng)脈粥樣硬化管腔病變處[37]。最近Borissoff等[38]表明循環(huán)中NETs的水平與動(dòng)脈粥樣硬化的相關(guān)性。2012年4月，D?ring等[2]在Circulation發(fā)表的文章中，闡述了動(dòng)脈粥樣硬化和自身免疫疾病之間的關(guān)系，文章指出：細(xì)胞外游離的DNA-蛋白質(zhì)復(fù)合物刺激漿細(xì)胞樣樹突狀細(xì)胞導(dǎo)致動(dòng)脈粥樣硬化的發(fā)生發(fā)展。D?ring等在同年6月又發(fā)表文章表明這種DNA-蛋白質(zhì)復(fù)合物通過誘導(dǎo)樹突細(xì)胞合成并釋放促動(dòng)脈粥樣硬化物質(zhì)干擾素γ增加斑塊負(fù)荷[3]。在ApoE基因缺陷小鼠，pDCs顯示加重動(dòng)脈粥樣硬化相關(guān)炎癥[39]。此外，Villanueva等[40]觀察到在炎癥過程中NETs對(duì)內(nèi)皮細(xì)胞的細(xì)胞毒作用。Warnatsch等[41]研究表明，膽固醇結(jié)晶誘導(dǎo)NETs生成，反過來(lái)又激活Th17細(xì)胞和巨噬細(xì)胞釋放IL-1β。血小板來(lái)源的趨化因子刺激NETs生成[42]。NETs可促進(jìn)局部的凝血活性通過借助血小板促進(jìn)凝血酶的生成機(jī)制[43]。另一方面，NETs可增強(qiáng)血小板活化，促進(jìn)動(dòng)脈粥樣硬化在小鼠的下肢深靜脈血栓形成[44]。

動(dòng)脈粥樣硬化對(duì)人類健康的威脅日益增大，其病理學(xué)研究主要集中在炎性細(xì)胞浸潤(rùn)及脂質(zhì)沉積，且斑塊局部有胞外誘捕網(wǎng)的形成，胞外誘捕網(wǎng)是炎性細(xì)胞激活后的結(jié)果，那么動(dòng)脈粥樣硬化中的胞外誘捕網(wǎng)形成可能與脂質(zhì)沉積發(fā)生相互作用，協(xié)同促進(jìn)動(dòng)脈粥樣硬化的發(fā)生發(fā)展。對(duì)胞外誘捕網(wǎng)的進(jìn)一步研究，有望為動(dòng)脈粥樣硬化的形成提供新的機(jī)制，同時(shí)也為動(dòng)脈粥硬化相關(guān)疾病提供新的診斷及治療思路。

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本文編輯：孫竹

R543.5

A

1674-4055(2017)05-0638-03

作者地址：1150000 哈爾濱,哈爾濱醫(yī)科大學(xué)附屬第二醫(yī)院心內(nèi)科

楊爽,E-mail:dryangshuang@163.com

10.3969/j.issn.1674-4055.2017.05.38