魚類血栓細胞免疫功能的研究現狀

高迎莉, 韓高尚, 姚海靜, 陳香凝, 周謙青

魚類血栓細胞免疫功能的研究現狀

高迎莉1, 2, 3, 韓高尚1, 姚海靜1, 陳香凝1, 2, 3, 周謙青1

(1. 江蘇海洋大學 海洋生命與水產學院, 江蘇 連云港 222000; 2. 江蘇省海洋生物技術重點實驗室, 江蘇 連云港 222000; 3. 江蘇省海洋生物資源與環境重點實驗室, 江蘇 連云港 222000)

血細胞研究是魚類基礎研究的重中之重。魚類血細胞的前期研究主要聚焦于淋巴細胞、巨噬細胞和嗜中性粒細胞等細胞的功能特性, 而忽略了血栓細胞。血栓細胞是低等脊椎動物具有的類似于哺乳動物血小板的一類細胞, 該細胞的經典功能是參與凝血和血栓形成。近年來的研究表明, 血栓細胞是一類特殊的免疫細胞, 在魚體免疫應答中發揮重要作用。作者將對魚類血栓細胞免疫功能相關的形態學、免疫學功能和研究前景等內容進行綜述, 為魚類血栓細胞功能的多樣性提供基礎資料。

形態學; 免疫功能; 血栓細胞; 魚類

1882年, Giulio[1]描述血液中一種新的“形態學成分”, 命名為血小板(Platelets), 并發現血小板在機體出血和血栓形成中發揮作用, 因此Giulio被稱為“血小板之父”。后來的研究發現, 哺乳動物無核的血小板來源于骨髓中的巨核細胞, 在非特異性和特異性免疫反應中發揮重要作用[2]; 低等脊椎動物如鳥類[3]、爬行類[4]、兩棲類[5]和魚類[6]中, 類似于血小板的細胞是有核的血栓細胞。近年來的研究表明, 血栓細胞參與魚類的非特異性免疫反應以及炎癥反應[7-9]。此外, 血栓細胞參與魚類的特異性免疫應答的研究引起眾多研究者的興趣[7-8, 10-12]。魚類的血栓細胞是否具有功能多樣性, 有待于進一步的研究。作者將對魚類血栓細胞免疫功能相關的形態學和細胞發生、免疫學功能等研究現狀進行綜述, 以全面闡述魚類血細胞的新功能, 為魚類血栓細胞功能的多樣性提供基礎資料。

1 血栓細胞的形態學特征

硬骨魚血液中, 血栓細胞在數量上僅次于紅細胞, 這些數量龐大的血栓細胞參與魚體的血栓形成和凝血過程[6]。血栓細胞成群分布, 呈淚滴型、紡錘型、卵圓型或孤核型等多種形態, 具有指狀突起、絲狀偽足、囊泡、液泡、微管系統、顆粒、線粒體、高爾基體以及核糖體等成分[7, 13-23]。Jagadeeswaran[24]等發現斑馬魚()血栓細胞具有開放的管狀系統, 血栓細胞活化后, 出現絲狀偽足, 細胞發生凝集反應。斑點叉尾鮰()血栓細胞呈梭狀, 胞內具有富含吞噬物質的液泡, 胞質中含有彌漫性的糖原顆粒[25](圖1a)。聚蓋魚() 血栓細胞有梭形和球形兩種, 細胞核有中心常染色質, 細胞質有液泡、管狀系統以及吞噬體[26](圖1b)。Azevedo[27]等的研究發現, 白紋笛鯛()血栓細胞被紅細胞圍著, 可形成多個胞質突起而呈現不規則細胞膜, 同時血栓細胞通過投影接近多個紅細胞的細胞膜; 血栓細胞核內的異染色質和常染色質中包含部分顆粒原纖維團塊及部分發育的液泡, 這些液泡有的位于胞質突起的頂端, 有的在細胞質中隨機分布。最新研究表明, 人類血小板的細胞膜具有偽足和突起, 細胞內部具有液泡、α-顆粒、致密顆粒和溶酶體顆粒、管狀系統、線粒體和自噬體。此外, 人類血小板的形態結構與年齡相關, 年齡增大, 細胞膜的規整性降低, 光滑性減少, 偽足和突起增多且更細長, 細胞內部的α-顆粒顯著減少[28]。綜上所述, 不同種魚類的血栓細胞在形態結構上存在差異, 但如突起、偽足、囊泡、溶酶體等跟免疫相關的形態結構均出現在血栓細胞中, 這為血栓細胞的功能多樣性奠定了結構基礎。而人類血小板的形態結構是伴隨時間發生變化, 因此不同魚類血栓細胞的結構差異是否與魚體的年齡、性別、健康狀態等相關, 仍需要進一步探究。

圖1 血栓細胞的超微結構觀察

a. Ultrastructure of thrombocytes in channel catfish. Fusiform thrombocyte with vacuoles, 1 of which contains phagocytosed material, bar=500 nm[25]; b. Ultrastructure of thrombocytes in fat snook. A fusiform thrombocyte to the left with its nucleus (N), to the right containing large phagosomes (fg), bar=2 μm[26]

細胞化學染色是以細胞形態學為基礎, 根據某一特定胞內組分的化學反應原理, 在顯微鏡下觀察細胞化學成分及其變化的顯微研究方法。常用的有酶類(酸性磷酸酶ACP、堿性磷酸酶ALP、過氧化物酶Peroxidase、酯酶等)、脂類(蘇丹黑B染色SBB)、糖原(過碘酸-希夫反應PAS)等細胞化學染色, 且常聯合吉姆薩Giemsa染色用于區別不同細胞亞群或者用于細胞功能的研究(圖2)。硬骨魚類種類不同, 血栓細胞的細胞化學特性有差別, 如莫瑞鱈魚(, Mitchell)[29](圖3)等魚類的血栓細胞呈現不同程度的細胞化學特性的相似性與差異性(表1)。除了藍旗金槍魚()[30], 所有魚類的血栓細胞都是PAS陽性, 糖原是中性粒細胞進行胞內殺傷以及清除外來物質時所需的能量來源, 這從形態學角度證明魚類血栓細胞的吞噬潛能。藍旗金槍魚是大洋洄游性魚類, 其血栓細胞的功能與其他魚類存在差異, 表明不同生境魚類的血栓細胞會進化出與其生境或者生理活動相匹配的功能, 藍旗金槍魚血栓細胞是否具有吞噬功能需要進一步的研究。人類血小板的吞噬溶酶體中含有一些酶類, 如ACP, 這是血小板具有吞噬能力的證據[31]。由表1可知, 除了莫瑞鱈魚、花斑裸鯉[32]和虹鱒[33], 其他魚類血栓細胞都是ACP陽性, 表明這些魚類的血栓細胞具有吞噬活性。但是, 莫瑞鱈魚、花斑裸鯉和虹鱒的血栓細胞超微結果顯示,細胞表面具有管狀系統, 胞內有吞噬小泡和吞噬物, 說明這3種魚類血栓細胞的吞噬活性是機械式而非酶活式。其他的細胞化學染色結果差別較大, 呈現明顯的種屬特異性, 如大蓋具脂鯉和條紋鯪脂鯉的ALP染色結果相反; 鏟吻油鯰[34]是蘇丹黑B陰性; 熱帶魚[35]是過氧化氫陰性, 但黃鲇魚[36]和大口黑鱸[37]是過氧化氫陽性; 羅非魚[38]僅僅檢測了糖原特性。魚類血栓細胞的形態學研究不僅可用于區分魚類的不同血細胞類群, 而且可以直接觀察血栓細胞的外部細胞膜、突起等結構以及內部細胞器等組成, 這為進一步研究血栓細胞的功能特性奠定結構基礎。此外, 細胞化學特性為魚類血栓細胞的種屬差異性、年齡相關性、健康狀態相關性、吞噬活性、免疫功能相關性等的研究提供直接證據。

圖2 魚類血栓細胞的形態圖

a. The thrombocytes of channel catfish (arrowhead) are smaller than the erythrocytes, with a spindle-shaped nucleus[25]; b. The thrombocyte of southern stingray (arrowhead)[23]; c. The thrombocyte of white sturgeon (arrowhead)[23]

圖3 莫瑞鱈魚血栓細胞的細胞化學染色

a. 過氧化氫染色; b. 酸性磷酸酶(ACP)染色; c. 堿性磷酸酶(ALP)染色; d. 氯乙酸AS萘酚酯酶(NCE)染色; e. 乙酸萘酯酶(NAE)染色; f.α-丁酸萘酯酶(NBE)染色; g. 蘇丹黑B(SBB)染色; h. 糖原(PAS)染色; i. β-葡糖醛酸糖苷酶染色。放大倍數×100, 標尺=10 μm[29]

a. peroxidase staining; b. acid phosphatase (ACP) staining; c. alkaline phosphatase (ALP) staining; d. naphthol AS chloroacetate esterase (NCE) staining; e. naphthyl acetate esterase (NAE) staining; f. α-naphthyl butyrate esterase (NBE) staining; g. Sudan black B (SBB) staining; h. periodic acid Shiff”s (PAS) staining; i. β-glucuronidase staining. Magnification × 100, Bar=10 μm[29]

表1 血栓細胞和血小板的細胞化學特性

續表

注: “+”. 陽性; “–”. 陰性; 鯉魚[8, 10](); 胭脂魚[17](); 條紋鯪脂鯉[25](); 大蓋具脂鯉[25](); 聚蓋魚[26](); 莫瑞鱈魚[29](, Mitchell); 藍鰭金槍魚[30](); 花斑裸鯉[32](); 虹鱒[33](); 鏟吻油鯰[34](); 熱帶魚[35](); 黃鲇魚[36](); 大口黑鱸[37](); 羅非魚[38]()

2 血栓細胞的免疫學功能

2.1 吞噬作用

吞噬作用是內吞作用的特殊形式, 包括顆粒的水泡內化, 形成內部囊泡即吞噬溶酶體, 是非特異性防御病原微生物的重要機制[39]。魚類專業的吞噬細胞有單核/巨噬細胞[40]、嗜中性粒細胞[41]和樹突狀細胞[42], 此外, 淋巴細胞也具有吞噬作用[43-45]。關于上述各類細胞吞噬功能的研究多采用細胞系培養或細胞分離方法, 將胞內寄生蟲、酵母菌、熒光微球和胞內寄生細菌等作為被吞噬的顆粒物, 應用細胞化學染色、光學顯微鏡、熒光顯微鏡、透射電鏡以及流式細胞術等技術, 證明細胞的吞噬作用。

近年來的研究結果表明, 魚類血栓細胞也具有吞噬作用, 但是血栓細胞在病原菌吞噬和清除上存在爭議, 這可能是實驗技術的不同造成的。細胞形態學結果顯示, 血栓細胞胞內存在具有吞噬能力的顆粒[46], 然而, 細胞化學分析表明, 血栓細胞ACP呈陰性[37]。但人類血小板吞噬體酶類鑒定結果表示, ACP陽性是吞噬體不可或缺的成分。因此, 這種不一致是由于研究方法的局限性, 還是由于魚類血栓細胞的細胞化學特殊性, 有待于進一步研究。脊椎動物的血小板吞噬作用具有4個特征, 且在魚類血栓細胞中也有體現: (1) 血栓細胞與顆粒性物質(非生物顆粒、病原體等)相互作用。鯉魚血栓細胞可以攝入并殺傷金黃色釀膿葡萄球菌()[46]以及大腸埃希氏菌()[8]; Burrows[47]等發現, 瘤棘鲆()血栓細胞能夠在體外主動吞噬碳顆粒, 具有較強的吞噬能力; 牙鲆()和鯉魚血栓細胞(圖4)能夠在體外吞噬熒光微球[8]; (2) 抗原刺激后, 血栓細胞胞質的溶酶體產物被釋放, 導致胞內殺菌作用。白紋笛鯛血栓細胞的超微結構顯示, 抗原刺激后, 胞內微管釋放空泡物質, 而該物質是血栓細胞吞噬作用的產物, 從細胞結構上印證血栓細胞的吞噬作用[27]; 鯉魚血栓細胞吞噬溶酶體融合實驗表明, 血栓細胞可以內化細菌且具有殺菌能力[8]; (3)吞噬作用的代謝產物能作為炎癥反應的媒介。Ferdous[3]研究了雞血栓細胞吞噬作用, 證明其代謝產物介導炎癥反應。而Nagasawa[12]等通過研究血栓細胞吞噬作用與白細胞分泌的激活因子之間的關系, 發現血栓細胞的吞噬活性會因白細胞分泌激活因子的增加而增加, 從而使其代謝產物在急性炎癥反應中發揮作用; (4)利用補體, 通過調理素作用增強吞噬消化功能。研究表明, 鯉魚血清對血栓細胞的吞噬活力起到增強的作用[8]。以上關于魚類血栓細胞吞噬作用研究主要集中于體外吞噬, 血栓細胞體內吞噬研究涉及較少, 研究方法單一, 缺乏多種方法的聯合使用以及方法間的相互佐證, 且血栓細胞吞噬作用介導的胞內殺傷作用等仍然不夠明確, 這在很大程度上限制對魚類血栓細胞抗菌功能的認知與探索。

圖4 鯉魚血栓細胞吞噬熒光微球和細菌

a.b. 血栓細胞吞噬熒光微球(X為被吞噬的微球); c. 血栓細胞通過延伸偽足吞噬細菌, 箭頭所示偽足, X.細菌; d. 小囊泡圍繞著被攝入的細菌, *. 囊泡, 標尺=1 μm[8]

a.b. Phagocytosis with fluorescent beads by thrombocytes (Ingested beads are indicated as X); c. Thrombocytes engulfed bacteria (X) via the extension of pseudopods (arrowhead); d. Small vesicles surrounding the internalized bacteria are indicated by asterisks (*), Bar=1 μm[8]

2.2 非特異性免疫反應

機體組織損傷后, 血小板/血栓細胞會參與機體的穩態以及血栓形成, 進而參與炎癥反應。血小板/血栓細胞會在第一時間聚集于損傷處, 除了聚集和參與炎癥反應, 血小板/血栓細胞表達和釋放有利于傷口組織修復的蛋白質和物質[48]。不同動物的血栓細胞分泌不同的細胞因子參與炎癥反應和傷口修復。鳥類血栓細胞產生一些生物活性物質, 即趨化因子(巨噬細胞炎癥蛋白-1β和一氧化氮)以及炎癥反應的媒介物質, 如誘導型一氧化氮合酶(iNOS)等; 雞血栓細胞能夠表達抗炎細胞因子(轉化生長因子TGF和IL-10)和前炎癥因子(IL-1β, IL-6, IL-8和 IL-12)以及利于傷口修復的細胞因子[3]。鯉魚和虹鱒血栓細胞能表達IL-1β、iNOS、腫瘤壞死因子TNFα、TGFβ和一些趨化因子[7-8]。

在哺乳動物中, 血小板可以表達一些非特異性免疫相關分子, 如Toll樣受體(Toll-like receptors, TLR)和抗原呈遞相關基因。鯉魚、大西洋鱈魚()、三刺魚()和虎河豚()的血栓細胞可以表達輔助分子(TLR4 interactor with leucine-rich repeats, TRIL)[9], 這表明魚類血栓細胞會像其他白細胞一樣, 在非特異性免疫反應中直接發揮作用。

2.3 特異性免疫反應

抗原呈遞是激活特異性免疫反應過程中的最后一個步驟, 主要經歷: (1) 攝取抗原; (2) 將抗原呈遞給特異性的胞內組分; (3) 將抗原肽釋放到抗原結合位點-主要組織性相容性復合體Ⅱ分子(MHC II)上; (4) 激活CD4輔助性T細胞。魚類的單核/巨噬細胞、樹突狀細胞和B淋巴細胞除了具有直接抗菌作用外, 還表達Ⅱ型組織相容性復合體, 且具有抗原處理和呈遞的分子機制。低等脊椎動物血栓細胞表面具有MHC Ⅱ分子, 如鳥類[49]、虹鱒和鯉魚[7-8, 10-11]的血栓細胞能夠表達MHC Ⅱ分子mRNA, 參與抗原呈遞過程。此外, 吞噬異物的血栓細胞以及具有抗原承載力的血栓細胞在鯉魚的頭腎和脾臟中都有所發現, 也說明血栓細胞具有運載抗原到淋巴組織并進入特異性免疫階段的能力[8]。以上研究僅局限于定量PCR的方法, 鑒定血栓細胞中免疫相關基因的表達水平變化, 缺少不同層次(基因水平、蛋白水平、細胞水平)的科學證據來論證血栓細胞的免疫功能。目前, 魚類血栓細胞非特異性和特異性免疫反應的分子水平研究與哺乳動物血小板還存在很大的差距。

3 血栓細胞的未來研究方向

魚類血栓細胞的免疫功能研究具有較大的發展空間。目前, 魚類血栓細胞的研究主要集中于魚類血栓細胞特性研究, 這將仍是魚類血栓細胞研究的方向。此外, 魚類血栓細胞特性是否和人類血小板類似, 與自身因子(年齡)等因素有關[28], 仍需深入研究。因此, 魚類血栓細胞特性與外界環境因素(棲息地、溶氧、pH等)以及內部因子(種類、年齡、性別、生理階段等)的關系將會是研究的重點。血栓細胞表面特異性標記物及受體[49-52]發掘, 可以研發血栓細胞的特異性單克隆抗體, 如虹鱒[7]、斑馬魚[53]、鯉魚[54]和牙鲆[55], 結合免疫磁珠、流式細胞術等方法, 提高魚類血栓細胞的分離純化效率[56-57]。利用分離純化的血栓細胞, 在前期研究的基礎上[7-12], 研究血栓細胞與其他免疫細胞的相互作用, 比如草魚血栓細胞與白細胞的作用[12], 血小板與樹突狀細胞的相互作用[58]; 借鑒血小板[59]和雞血栓細胞[60]的研究方法, 開展魚類血栓細胞的組學研究和免疫調控網絡分析, 將會是未來研究的熱點。此外, 樹蛙()血栓細胞系已經建立[5], 能否建立魚類血栓細胞的細胞系, 開展胞外囊泡[61]等熱點問題研究, 仍需要進一步探究。

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Current research on the immune function of thrombocytes in fish

GAO Ying-li1, 2, 3, HAN Gao-shang1, YAO Hai-jing1, CHEN Xiang-ning1, 2, 3, ZHOU Qian-qing1

(1. College of Marine Life and Fisheries, Jiangsu Oean University, Lianyungang, 222000, China; 2. Jiangsu Key Laboratory of Marine Biotechnology, Lianyungang 222000, China; 3. Jiangsu Key Laboratory of Marine Bioresources and Environment, Lianyungang 222000, China)

The research on blood cells is very crucial to the research on fish. Research is basically focused on the immune functions of lymphocytes, macrophages, and neutrophils. There is limited focus on thrombocytes. Platelets in mammals and thrombocytes in lower vertebrates are implicated in thrombotic and hemostatic processes. The thrombocytes are regarded as special immune cells that play important roles in the immune response of fish based on current reports. This paper systemically reviews the immune-associated morphological features, immune functions, and future research perspectives on thrombocytes, with the aim of providing basic insight into the multi- functional nature of thrombocytes.

Morphology; immune function; thrombocyte; fish

Nov. 6, 2019

Q-1

A

1000-3096(2021)01-0120-09

10.11759/hykx20191106001

2019-11-06;

2020-05-25

江蘇省自然科學基金青年基金項目(BK20170450); 江蘇省省政策引導類計劃-蘇北科技專項項目(SZ-LYG2017020); 江蘇省高等學校自然科學研究面上項目(17KJB240001); 江蘇省生物技術重點實驗室開放基金項目(HS2017003)和江蘇省海洋資源與環境重點實驗室開放基金項目(CXKT20180112); 江蘇省大學生創新創業項目(SY201811641105003)

[Natural Science Foundation of Jiangsu Province, No. BK20170450; Policy Guidance Program of Jiangsu Province, No. SZ-LYG2017020; Natural Science Foundation of the Jiangsu Higher Education Institutions of China, No. 17KJB240001; The Open Research Fund of Jiangsu Key Laboratory of Marine Biotechnology, No.HS2017003; The Open Research Fund of Jiangsu Key Laboratory of Marine Bioresources and Environment, No. CXKT20180112; Student Innovation and Entrepreneurship Program of Jiangsu Province, No. SY201811641105003]

高迎莉(1988- ), 女, 江蘇徐州人, 講師, 博士, 主要從事水產動物病害與免疫學研究, E-mail: yingligao0127@126.com

(本文編輯: 譚雪靜)