魚類免疫應答機制研究進展

白姍姍，賈智英，石連玉

（1.中國水產科學研究院黑龍江水產研究所，黑龍江 哈爾濱 150070；2.上海海洋大學水產與生命學院，上海 201306）

魚類免疫應答機制研究進展

白姍姍1,2，賈智英1，石連玉1

（1.中國水產科學研究院黑龍江水產研究所，黑龍江 哈爾濱 150070；2.上海海洋大學水產與生命學院，上海 201306）

魚類免疫應答可以分為固有免疫和適應性免疫，但固有免疫發揮主要作用。固有免疫對病原體的識別是通過模式識別受體PRR與病原相關分子模式PAMP的相互結合實現，這與哺乳類相似。但為適應水生生活，魚類固有免疫對PAMP的識別范圍更廣，免疫應答的啟動條件更低。固有免疫的效應細胞主要是單核/巨噬細胞、嗜中性粒細胞、自然殺傷細胞等，具有吞噬和殺傷功能，還可分泌多種免疫相關的細胞因子，介導發生炎癥反應。適應性免疫中，T淋巴細胞通過抗原提呈細胞分解吸收抗原，由主要組織相容性復合物（MHC）類分子遞送到細胞表面才能識別。B淋巴細胞分泌產生以免疫球蛋白IgM為主的抗體分子，而發揮抗體中和作用及免疫調理作用的IgG在魚類中比較少見，說明魚類抗體的免疫功能還處于較低水平。本文綜述了近二十年內魚類免疫應答機制的相關研究進展，為進一步了解魚類免疫應答機制提供參考。

魚類；適應性免疫；固有免疫；免疫應答

我國魚類養殖歷史悠久，市場需求巨大，養殖過程中的病害問題也相當突出。每年由各類真菌、細菌及病毒誘發的疾病，給我國魚類養殖業造成巨大的經濟損失，因此研究魚類免疫應答尤為重要。增強魚類免疫應答水平，以提高機體抗病能力是研究魚類抗病免疫的重中之重。免疫應答是指機體對于異己成分或者變異的自體成分做出的防御反應。而免疫應答的一般過程包括抗原呈遞與識別階段、活化增殖和分化階段、效應階段。魚類免疫學研究證明，與哺乳動物等高等脊椎動物一樣，魚類免疫存在適應性（特異性）免疫和固有（非特異性）免疫。但是，魚類是變溫水生動物，免疫應答過程中固有免疫應答機制起主要作用。

1 固有免疫應答

魚類固有免疫應答具有種系內穩定遺傳、沒有特異性、免疫識別廣泛的特點，與抗原的初次接觸即產生效應，不具有二次免疫功能，協助并參與適應性免疫應答。

1.1 防御屏障的作用

由皮膚、鱗片、粘膜表皮層構成魚類的基礎屏障是魚類的第一線防御。魚類表皮黏液中含有溶菌酶和糖蛋白等，能抑制細菌微生物的生長或使其失活。溶菌酶廣泛存在于淡、海水魚類的皮膚黏液、血清、淋巴組織和組織器官中[1]，可以分解細菌細胞壁的肽聚糖。魚類黏液中存在特異性抗體（尤其是IgM）[2]，可直接對入侵的病原體發揮免疫作用。

1.2 固有免疫應答的識別機制

免疫應答離不開對抗原的識別。魚類固有免疫對抗原的識別是通過模式識別分子和模式識別受體（PRR）識別病原體相關分子模式（PAMP）實現的。PRR與病原體表面的PAMP相互識別和作用是啟動固有免疫應答的關鍵。值得注意的是，PAMP很少是蛋白質，蛋白質類病原體主要為淋巴細胞識別，誘發適應性免疫應答[3]。

模式識別受體（PRR）包括吞噬性受體（可溶性受體）和信號受體（膜結合受體）。已證實，魚類的前者包括C型凝集素受體（CLR）、清道夫受體（SR），后者則包括 Toll樣受體（TLR）、NOD樣受體（NLR）、RIG樣受體（RLR）。CLR主要識別糖類抗原，介導并調節細胞因子的產生、上調單核/巨噬細胞（Mo/MΦ）[4,5]，并觸發激活Th細胞的抗真菌免疫應答[6]。硬骨魚類尼羅羅非魚Oreochromis niloticus的CLR與哺乳類NK細胞受體同源[7]。SR主要有SRA和SRB兩種，SRB能與革蘭氏菌結合[8]。SRA參與細菌及RNA病毒引發的免疫應答[9,10]。TLR是細胞膜表面識別受體，可以直接特異性結合病原體結構[11]，也可以激發NF-κB信號通路，而NF-κB通路是先天免疫系統中細胞信號通路激活的核心[12]。TLR信號轉導途徑為MyD88依賴途徑和非MyD88依賴途徑，分別參與炎癥反應和抗病毒效應[13,14]。目前在魚類中發現了十幾種TLR，其中TLR2、TLR5、TLR9可以專門識別細菌的PAMPs[15,16]。TLR4參與細菌和病毒引發的免疫應答[17,18]。對魚類參與介導炎癥級聯反應的NLR類型知之甚少，日本學者認為LPS可能是NLR介導炎癥反應的配體[19]。有學者推測，NLR在細胞調控、細胞凋亡及免疫應答中有重要作用，硬骨魚類中普遍存在保守的NLR基因序列[20]。RLR屬于I型干擾素誘導蛋白[21]，魚類的RLR對胞內、外的各種病毒均有識別功能，誘發IFN信號通路以抵抗病毒感染[22,23]。

模式識別分子（PRM）參與炎癥反應中病原體的清除。魚類PRM主要包括急性時相蛋白C反應蛋白（CRP）、抗菌/通透性蛋白（BPI）和脂多糖結合蛋白（LBP）。CRP在炎癥信號及IL-6的激發下由肝臟產生，對入侵的異己成分做出免疫應答[24]。因CRP參與補體的激活，常被作為炎癥反應的生物標志物[25]。魚感染嗜水氣單胞菌Aeromonas hydrophila后，血清中CRP含量增加[26,27]。哺乳動物在炎癥反應和組織壞死的早期CRP含量也增加，這一現象在尼羅羅非魚中已被證實[28]。部分硬骨魚類中，證實了BPI和LBP參與并誘導急性時相蛋白對細菌疾病的免疫應答[29,30]。

1.3 固有免疫的效應細胞

固有免疫的效應細胞主要有單核/巨噬細胞、嗜中性粒細胞、自然殺傷（Natural killer,NK）等，具有吞噬和殺傷功能，也分泌免疫相關的細胞因子，介導炎癥反應的發生。

單核/巨噬細胞與中性粒細胞是魚類中兩大類重要的吞噬細胞[31]。單核細胞遷移出血管至各組織中，發育為巨噬細胞。魚類的單核細胞與哺乳動物相似。硬骨魚類的細胞因子如IL-4、IL-13及γ-干擾素與巨噬細胞激活與分化有關[32,33]。單核巨噬細胞不僅在固有免疫中發揮吞噬作用，在適應性免疫中作為抗原提呈細胞APCs發揮作用。巨噬細胞可以被多種細胞因子（IL-1、IFN-γ等）活化，直接分泌殺傷物質直接殺死靶細胞，還可以分泌如IFN-γ、IL-1、TNF-α等細胞因子增強自身殺傷能力。魚類的IFNγ、IL-4、IL-13等細胞因子參與巨噬細胞極化為M1、M2類巨噬細胞的過程，也證明魚類中同樣存在Th1和Th2類免疫反應[32]。

嗜中性粒細胞是魚類固有免疫的重要細胞，魚類嗜中性粒細胞與哺乳動物嗜中性粒細胞的功能相似[34,35]，具有活躍的吞噬和殺傷功能，但其吞噬能力一般比單核/巨噬細胞弱。中性粒細胞胞外誘捕網（Neutrophil extracellular traps,NETs）由 DNA和抗菌蛋白組成，在固有免疫應答中阻攔并消除病原體，目前已經在鯉Cyprinus carpio中發現，是固有免疫的重要部分[36]。NK細胞因其非特異的細胞毒殺傷作用而得名。哺乳動物的NK細胞一般依靠IL-2和IL-12等細胞因子激活。這些細胞因子能直接影響NK細胞的遷移速率，促進NK細胞的增殖[37]。NK細胞通過釋放NK細胞毒因子（NKCF）、TNF-α及干擾素IFN-γ等殺傷介質和靶細胞。魚類的NK細胞包括非特異性細胞毒性細胞（NCCs）和NK細胞[38]。NCC細胞是源于器官的細胞，NK細胞來源于外周血淋巴細胞（PBL）[39]。NK細胞清除“異己”成分不需要預先激活，可以直接殺死病毒感染的細胞[40]。溫度影響自然殺傷細胞的殺傷作用及吞噬細胞等的吞噬作用[41]，這可能是某些魚類疾病發生時具有溫度依賴性的原因之一。而魚類中還存在自然殺傷細胞增強因子（NKEF），可增強魚類免疫病菌和病毒的免疫應答水平，是巨噬細胞和細胞毒性細胞在固有免疫應答過程中的溝通橋梁[42,43]。

1.4 固有有免疫的效應分子

大多數魚類的抗菌肽（AMPs）具有直接抗菌功能[44]，如溶菌酶（Lysozyme）可使細菌溶解[45]并激活補體系統，促進吞噬細胞的吞噬作用，防御素（Defense）能直接殺傷細菌活性[46,47]。

補體（Complement,C）具有調理、吞噬、炎癥介導、補體防御和清除病原體的作用[48]。補體活化途徑包括經典途徑、凝集素途徑和旁路途徑[49]。與哺乳類相比，魚類的補體存在多種亞型，可以識別更多的外源細胞表面[50]。C3是魚類補體系統的主要成分，可以調理中性粒細胞的吞噬作用[51]。無頜魚類的C3只能通過替代途徑激活，促進細胞吞噬[52]。補體C5介導炎癥反應中靶細胞溶解過程[53]。補體C6是免疫反應的重要感受器和效應器，參與消除感染細胞的過程[54]。補體C7在補體激活及效應過程中，作用于靶細胞膜上，發揮殺傷作用[55]，屬于效應分子。由于補體經典途徑的激活需要抗原抗體復合物（APC）的出現，所以補體也參與調節T細胞介導的適應性免疫應答[56]。

細胞因子是一類由免疫細胞和非特異性免疫細胞合成或分泌的小分子多肽物質，僅在機體進入免疫應答階段大量分泌[57]。魚類中已鑒別出的細胞因子有白細胞介素（Interleukin,IL）、趨化因子（Chemokine）、干擾素（IFN）、轉化生長因子（TGF-β）。

目前已經報道的魚類白細胞介素中IL-1β、IL-6、TNF-α具有促炎作用，IL-10具有抗炎作用，IL-21參與T細胞增殖/分化，IL-12刺激NK細胞激活[58,59]。由此可見，白細介素在魚類固有免疫和適應性免疫中均有不可替代的作用。IL-2和IL-6優先驅動巨噬細胞分化[60]。值得注意的是，IL-2可以誘導激活T細胞、B細胞和NK細胞分泌IFN-γ[61]，促進Th細胞分化并分泌IFN-γ介導細胞免疫[62]。在金頭鯛Sparus aurata發現的白細胞介素IL-6對TNF-α的活化作用由NF-κB、p38 MAPK和JNK信號轉導通路介導[63]。TNF可以誘發香魚Plecoglossus altivelis腎臟呼吸爆發，這與哺乳類相似[64]。與細菌相比，病毒可以更高更快地誘導TNF-α的產生[65]。

趨化因子（Chemokine）家族有四個亞族：C-X-C亞族、C-C亞族、C-X3-C亞族和C亞族。前3個亞族在魚類中均已發現[66]，魚類的趨化因子家族與哺乳類具有同源性，但是功能有所差別[67,68]。趨化因子可使趨化白細胞參與炎癥反應[69]。C-C趨化因子是趨化因子的最大亞家族，是先天免疫系統的重要組成部分，具有招募白細胞和增強固有免疫應答的功能[70]。在虹鱒Oncorhynchus mykiss中發現Fractalkine類似物，屬于C-X3-C趨化因子，對單核細胞和T淋巴細胞有趨化作用[71]。鯉C-X-C亞族對中性粒細胞、吞噬細胞有趨化作用[72]。魚類趨化因子C亞族的報道較少。

干擾素是一種能夠誘導多種細胞因子的多基因家族。根據結構和功能，IFN可分為三類：Ⅰ型、Ⅱ型和Ⅲ型。Ⅰ型和Ⅲ型干擾素的表達都與干擾素調節因子（IRF）以及核因子（NF-κB）有關[73]。I型干擾素斑馬魚Brachydanio rerio幼魚感染神經壞死病毒（NNV）的免疫至關重要[74]。轉化生長因子（TGF-β）家族在鮭鱒中已經成功分離，推測其可能與魚類病毒免疫相關[75]。

2 適應性免疫應答

2.1 淋巴細胞抗原識別、提呈及激活

T淋巴細胞通過抗原提呈細胞（APC）分解抗原，由MHC類分子遞送到細胞表面才能識別?？乖岢始毎饕袠渫粻罴毎?、B淋巴細胞和巨噬細胞，抗原提呈分子主要有MHC I類分子、MHC II類分子及T細胞表面受體（TCR）。其他抗原提呈輔助分子如 CD3、CD4、CD8等都與 T 細胞表面受體（TCR）的識別有關[76]。哺乳動物的T淋巴細胞表面分子標記CD4和CD8將T細胞分為輔助性T細胞和細胞毒性T細胞兩種主要亞群，魚類也有這兩種分子標記。CD4/CD8淋巴細胞同時出現在胸腺中，說明硬骨魚類的胸腺是T淋巴細胞發育的器官[77,78]。CD8和CD4分子分別通過與自身MHCⅠ類分子和Ⅱ類分子的恒定區結合，加強T細胞與APC或靶細胞的相互作用以及TCR-CD3信號的轉導。CD8+細胞毒性T淋巴細胞提呈MHC I類分子，識別內源性抗原。CD4+Th細胞提呈MHCII類分子，識別外源性抗原[79]。CD4+T細胞可以誘導產生二次免疫抗體[80]。參與T細胞活化的主要蛋白激酶和蛋白磷酸酶，通常前者在信號轉導的上游發揮作用，后者在下游發揮作用，并直接作用于轉錄因子[3]。IL-2、核轉錄因子NF-κB的活化和表達在T細胞的活化中發揮重要作用[81]。T細胞介導MHC遞呈的抗原信號后需要CD28或APC表面的配體分子結合才能被真正激活。目前僅在少數硬骨魚類中報道過CD28分子，對其具體生物學功能還知之甚少[82]。

B細胞對抗原的識別通過B細胞抗原受體BCR實現，可以識別多種抗原，不存在MHC類分子的限制條件。BCR是一種膜型免疫球蛋白mIg。輔助作用的受體有CD19、CD21、CD81等，在魚類中研究較少。鯉科魚類中IL-4、IL-13可以促進B淋巴細胞增殖和IgM的分泌[83]。IL-10與B淋巴細胞的活化有關，B淋巴細胞活化因子BAFF可以刺激IL-10和NF-κB的轉錄和表達[84]。在哺乳類中參與B淋巴細胞活化及增殖的細胞因子大部分是由Th細胞分泌，這一觀點還未在魚類中廣泛證實。

2.2 適應性免疫應答的效應機制

2.2.1 抗體的效應功能

B淋巴細胞激活分化為漿細胞后，分泌產生以免疫球蛋白IgM為主的抗體分子?？贵w結合抗原，激活補體經典途徑，形成攻膜復合物使靶細胞迅速裂解，現已證實魚類的IgM和補體C在免疫過程存在共同變化的特點[85]。免疫球蛋白主要存在于血漿中，也見于其他體液、組織和一些分泌液中。魚類有多種免疫球蛋白，如IgD、IgZ、IgT、IgR等。在哺乳類中發揮抗體中和作用及免疫調理作用的IgG在魚類中比較少見。免疫球蛋白M（IgM）主要由脾臟和淋巴結中漿細胞分泌合成，相對分子質量最大，幾乎存在于所有脊椎動物中[86]。胸腺是魚類重要的免疫器官，IgM的表達量較低。這可能是魚類的胸腺隨著生長而退化的結果[87]。外殼蛋白VP7與草魚Ctenopharyngodon idellus血清IgM特異性結合可免疫草魚呼腸孤病毒感染[88]。IgM廣泛分布于魚類的免疫器官如頭腎、脾臟中，而IgD、IgZ的分布范圍明顯較小[89]。IgZ、IgT、IgR主要在體表和腸道等黏膜系統發揮免疫作用[90]。虹鱒中存在一種專門B淋巴細胞分泌IgT，而IgM陽性B淋巴細胞卻不能分泌IgT[91]，這可能是因為B淋巴細胞在粘膜組織和非粘膜組織中發揮的免疫應答功能不同[92]。

2.2.2 T細胞介導的效應機制

在哺乳動物中，細胞毒性T細胞CTLs殺傷靶細胞的作用必須有Th細胞分泌的IL-2細胞因子的參與才能完成。CTLs首先通過MHC I類分子與靶細胞結合并釋放穿孔素殺傷靶細胞，該過程依賴Mg2+、Ca2+離子存在[93]。細胞毒性T細胞分泌TNF-α可以與靶細胞表面相應受體結合發揮細胞毒作用并殺死靶細胞[94]。魚類的細胞毒性T細胞參與病毒誘發免疫應答的功能和哺乳類相似，對CD8、TCR、MHC I類子等與CTLs免疫相關分子的mRNA表達譜的研究，證實了CTLs的抗病毒免疫功能[95]。

3 小結

雖然魚類是水生低等脊椎動物，但同樣具備固有免疫和適應性免疫。與哺乳類相比，魚類適應性免疫的進化程度較低，主要是因為免疫球蛋白的類型較少，再次免疫的記憶程度較低。多數魚類的抗體類型單一，有可能開發出其他潛在的免疫應答策略。魚類某些固有免疫可能優于哺乳類，如多種形式的溶菌酶、補體亞型、C反應蛋白及急性時相蛋白等，大大增加了固有免疫對抗原的識別能力，提高了免疫應答水平。但是魚類的免疫應答水平受外界環境影響巨大，尤其是溫度的變化，直接影響T細胞和補體等免疫成分的生物活性。魚類種類和品種多樣，免疫應答的種間差異很大，免疫應答作用機理也不能一概而論。隨著現代生物技術，尤其是基因分析、蛋白表達等技術的迅速發展，魚類免疫研究應從基因、蛋白、細胞水平進一步細致、深化魚類免疫應答機制的研究，盡早形成一套完善的魚類免疫應答機制理論。

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Research Progress of Immune Response Mechanisms in Fish

BAI Shan-shan1,2,JIA Zhi-ying1,SHI Lian-yu1
（1.Heilongjiang River Fisheries Research Institute,Chinese Academy of Fishery Sciences,Harbin 150070,China;2.College of Fisheries and Life Sciences,Shanghai Ocean University,Shanghai 201306,China）

Like mammals,fish immune response can be divided into innate immunity,which is primarily realized by identifying pathogens through the interactive combination of pattern recognition receptor（PRR）and pathogen-associated molecular pattern（PAMP）,and adaptive immunity.In order to adapt to aquatic life,however,fish innate immunity has a broader recognition scope on PAMP to launch immune response under low conditions.Effect cells of innate immunity are mainly comprised of mononuclear/macrophage,neutrophilic granulocyte and natural killer cells and others showing functions of phagocytosis and killing and secreting multiple immune-associated cytokines and mediate in inflammatory response.In the adaptive immunity,T lymphocytes absorb antigens through decomposition of antigen presenting cells and identifiation by transmitting to the surface by main histocompatibility complex（MHC）molecules,indicating the lower immunity under the limitation of MHC molecules,compared to the mammals.In the mammals,B lymphocytes generate antibody modules by the primary immune globulin IgM while the fish antibody modules are derived from the rare IgG,indicating that comparing with mammals,immunologic functions of fish antibody are still kept in the low level.The relevant articles of fish immune response mechanism published in the past 20 years are summarized,aiming to further know about the research progress of fish immune response mechanism.

fish;adaptive immunity;innate immunity;immune response

S917

A

1005-3832（2017）04-0059-09

2016-11-09

黑龍江所基本業務費(HSY201401)；國家“十二五”科技支撐計劃項目(2012BAD26B01)；國家大宗淡水魚類產業技術體系北方鯉魚種質資源與育種項目(CARS-46-02).

白姍姍（1993-），女，碩士研究生，研究方向：水產遺傳育種.E-mail:bss140101017@163.com

石連玉（1960-），男，研究員，從事魚類遺傳育種研究.E-mail:sly2552@yahoo.com.cn