甄夢(mèng)曉， 邢 婧， 繩秀珍， 唐小千， 戰(zhàn)文斌

(中國海洋大學(xué)水產(chǎn)學(xué)院水產(chǎn)動(dòng)物病害與免疫學(xué)研究室，山東 青島 266003)

遲緩愛德華氏菌感染和疫苗免疫后牙鲆的血細(xì)胞數(shù)量變化*

甄夢(mèng)曉， 邢 婧**， 繩秀珍， 唐小千， 戰(zhàn)文斌

(中國海洋大學(xué)水產(chǎn)學(xué)院水產(chǎn)動(dòng)物病害與免疫學(xué)研究室，山東 青島 266003)

重組表達(dá)了牙鲆(Paralichthysolivaceus)T淋巴細(xì)胞表面標(biāo)志分子CD3的蛋白，制備出其兔多克隆抗體(多抗)，采用免疫雙熒光流式細(xì)胞術(shù)，觀察外周血白細(xì)胞中的T、B淋巴細(xì)胞并計(jì)算其比例。分別向牙鲆腹腔內(nèi)注射107CFU/mL的遲緩愛德華氏菌和其滅活疫苗， 于0、1、3、5、7、9、14、21和28天后抽取外周血，用抗紅細(xì)胞單抗的流式細(xì)胞術(shù)測(cè)定紅細(xì)胞和白細(xì)胞的比例和濃度，提取外周血白細(xì)胞，用流式細(xì)胞術(shù)測(cè)定T、B淋巴細(xì)胞的比例。研究顯示，CD3多抗和IgM單抗分別識(shí)別T、B淋巴細(xì)胞，二抗體無交叉反應(yīng)，白細(xì)胞中T細(xì)胞占(7.49±1.5)%、B細(xì)胞占(14.64±1.8)%。感染和免疫后，牙鲆外周血紅細(xì)胞濃度變化不顯著，為(2.9±0.45)×109cells/mL。感染組，白細(xì)胞濃度第3天顯著上升，第14天達(dá)到最大值(4.7±1.7)×108cells/mL；T細(xì)胞比例第1天顯著上升，第9天達(dá)到最大值(24.3±1.28)%；B細(xì)胞比例第1天顯著升高，第21天達(dá)到最大值(40.9±1.52)%。免疫組，白細(xì)胞濃度第3天顯著上升，第14天達(dá)最大值(7.1±1.8)×108cells/mL；T細(xì)胞比例第5天顯著上升，第9天達(dá)最大值(20.5±1.12)%；B細(xì)胞比例第1天顯著上升，第21天達(dá)到最大值(39.3±1.55)%。對(duì)照組，白細(xì)胞濃度為(6.9±1.6)×107cells/mL，T細(xì)胞比例為(8.05±1.38)%，B細(xì)胞比例為(13.77±1.56)%。研究結(jié)果表明，感染和免疫后牙鲆白細(xì)胞濃度、T、B細(xì)胞的比例均顯著升高，感染組T、B細(xì)胞比例顯著高于免疫組。本研究結(jié)果為血細(xì)胞作為牙鲆健康評(píng)估指標(biāo)提供了數(shù)據(jù)資料。

牙鲆；感染；免疫；遲緩愛德華氏菌；T淋巴細(xì)胞；B淋巴細(xì)胞

牙鲆(Paralichthysolivaceus)血細(xì)胞直接參與機(jī)體非特異性免疫和特異性免疫應(yīng)答，在機(jī)體疾病的防御和防治中發(fā)揮著重要作用[1-2]。尤其外周血中的白細(xì)胞或T、B淋巴細(xì)胞的數(shù)量變化，能夠反映機(jī)體的整體免疫狀態(tài)[3]，研究表明，在魚體被細(xì)菌感染引發(fā)炎癥反應(yīng)時(shí)，白細(xì)胞數(shù)量會(huì)顯著升高[4-5]。因此，在魚體被病原侵染后，監(jiān)測(cè)白細(xì)胞和淋巴細(xì)胞的數(shù)量變化可作為研究其機(jī)體免疫動(dòng)態(tài)及感染進(jìn)程的方法之一[6-7]。

關(guān)于魚類血細(xì)胞數(shù)量的研究，經(jīng)常采用Giemsa或Wright染色方法，分類計(jì)數(shù)各類細(xì)胞數(shù)量[8]或血細(xì)胞分析儀直接計(jì)數(shù)[9]。由于T、B淋巴細(xì)胞表面存在特異性的標(biāo)志分子，T細(xì)胞表面受體(TCR)和表面分子CD3是T細(xì)胞進(jìn)行抗原識(shí)別最重要的一對(duì)復(fù)合體[10-14]，哺乳動(dòng)物對(duì)T細(xì)胞的檢測(cè)是通過抗CD3分子的單抗對(duì)T淋巴細(xì)胞進(jìn)行分類計(jì)數(shù)；B細(xì)胞的表面受體(BCR)由細(xì)胞膜免疫球蛋白(SmIg)和Igα(CD79a)/Igβ(CD79b)異源二聚體組成[15-18]，通常利用抗IgM分子的單抗和抗CD79a/CD79b分子的抗體來檢測(cè)B淋巴細(xì)胞的數(shù)量。利用淋巴細(xì)胞表面標(biāo)志物的抗體對(duì)相應(yīng)淋巴細(xì)胞類型進(jìn)行標(biāo)記，應(yīng)用流式細(xì)胞術(shù)測(cè)定淋巴細(xì)胞比例[6,19,21]，具有特異性高，檢測(cè)方便等優(yōu)勢(shì)。牙鲆中，應(yīng)用鼠抗牙鲆IgM單抗標(biāo)記B淋巴細(xì)胞測(cè)定牙鲆外周血中IgM+B細(xì)胞的比例[19]；圓鰭魚中，利用IgM單抗測(cè)定外周血中IgM+B細(xì)胞的比例約為41%[20]；河豚中，利用鼠抗CD8α的多抗測(cè)定河豚外周血中CD8α+T細(xì)胞的比例[21]。

本實(shí)驗(yàn)室前期已研制出抗牙鲆IgM的單抗[19]和抗牙鲆紅細(xì)胞單抗[22]。本文研制出兔抗CD3多抗，采用腹腔注射遲緩愛德華氏菌(Edwardsiellatarda)和其滅活疫苗，應(yīng)用抗牙鲆紅細(xì)胞單抗的流式細(xì)胞術(shù)測(cè)定牙鲆外周血中紅細(xì)胞數(shù)量比例，并計(jì)算白細(xì)胞的數(shù)量；應(yīng)用兔抗牙鲆CD3多抗和鼠抗牙鲆IgM單抗的流式細(xì)胞術(shù)檢測(cè)白細(xì)胞中T、B淋巴細(xì)胞比例變化，以期為牙鲆健康評(píng)估指標(biāo)的研究提供數(shù)據(jù)資料。

1 材料與方法

1.1 實(shí)驗(yàn)動(dòng)物

牙鲆購自于山東日照某養(yǎng)殖場(chǎng)，體長為15 cm左右，體重為20～50 g，750尾，實(shí)驗(yàn)前于水箱中暫養(yǎng)1周，水溫20～23 ℃，連續(xù)充氣。

新西蘭兔購于青島某實(shí)驗(yàn)動(dòng)物中心，實(shí)驗(yàn)前于實(shí)驗(yàn)室養(yǎng)殖兔房室溫暫養(yǎng)1～2周。

1.2 牙鲆外周血的抽取、白細(xì)胞及cDNA的提取

采用尾靜脈抽血，抗凝劑(50 mL RPMI-1640，加入0.0067 g肝素鈉和0.5 g 牛血清白蛋白)與外周血按2∶1比例混合，置于10 mL離心管中。用微量移液器吸取10 μL血細(xì)胞懸液，用PBS稀釋200倍，用于吉姆薩染色和流式細(xì)胞術(shù)。

采用percoll梯度離心法提取牙鲆外周血白細(xì)胞。抽取的外周血樣品靜置1 h后，以100g4 ℃離心10 min，收集上清；用過濾除菌的新生牛血清潤洗無菌離心管壁，用移液器吸去血清，緩慢加入3 mL的1.070 g/cm3的Percoll應(yīng)用液，再用移液器沿管壁緩慢加入3 mL的1.020 g/cm3的Percoll應(yīng)用液，制備Percoll不連續(xù)梯度，再用移液器緩慢加入3 mL收集的外周血離心上清，以840g轉(zhuǎn)速于4 ℃離心30 min；收集Percoll梯度界面上的細(xì)胞，用無菌PBS重懸，并以680g轉(zhuǎn)速于4 ℃離心6 min，重復(fù)洗3次，最后收集的沉淀即為白細(xì)胞。

此時(shí)，一部分白細(xì)胞用于提取mRNA，反轉(zhuǎn)錄得到cDNA，作為CD3基因擴(kuò)增的模板；將另一部分白細(xì)胞重懸，濃度調(diào)整至106cells/mL,用于吉姆薩染色和流式細(xì)胞術(shù)。

1.3 牙鲆CD3胞外區(qū)蛋白重組表達(dá)和兔多抗的制備

根據(jù)GenBank公布的牙鲆CD3ε序列(GenBank: AB044572.1)，對(duì)應(yīng)其胞外區(qū)序列，設(shè)計(jì)特異性引物[13](見表1)，以得到的cDNA為模板，PCR擴(kuò)增出大小為534 bp的單一目的條帶，PCR產(chǎn)物經(jīng)回收純化，送檢測(cè)序。將測(cè)序正確的目的基因連接到pET-30a表達(dá)載體上，轉(zhuǎn)化到感受態(tài)大腸桿菌BL21中，得到重組表達(dá)菌。將重組表達(dá)菌接種到含0.1% 氨芐抗生素的500 mL LB液體培養(yǎng)基中，37 ℃恒溫培養(yǎng)4～6 h，然后加入終濃度為1.0 mmol/L IPTG誘導(dǎo)表達(dá)12 h后，離心，收集菌體，SDS-PAGE驗(yàn)證CD3重組表達(dá)菌的誘導(dǎo)表達(dá)結(jié)果；再用含尿素2 mol/mL的50 mL Binding buffer(2 mol/L 尿素，0.5 mol/L NaCl，20 mmol/L Na3PO4，30 mmol/L 咪唑，調(diào)整pH至7.4)重懸菌體，于冰浴中超聲破碎30 min；最后用鎳瓊脂糖親和層析柱(HisTrapTMHP 5mL購于GE生命科學(xué)公司)純化，得到重組蛋白。將蛋白透析并凍干濃縮，PBS溶解，利用Bradford蛋白濃度測(cè)定試劑盒(購于索萊寶公司)測(cè)定濃度，調(diào)至1 mg/mL，用于免疫新西蘭兔。

兔多抗的制備：第一周基礎(chǔ)免疫，純化的CD3重組蛋白樣品與弗氏完全佐劑1∶1混勻，皮下多點(diǎn)注射新西蘭兔，每點(diǎn)0.2 mL；第三周蛋白懸液與弗氏不完全佐劑1∶1混勻加強(qiáng)免疫；接下來2周再加強(qiáng)免疫2次，耳緣靜脈注射蛋白懸液0.2 mL，無佐劑；第六周對(duì)新西蘭兔進(jìn)行心臟采血，血液于室溫放置2 h，4 ℃過夜，以8 000g離心10 min，取上清，分裝后-80 ℃保存，備用。通過ELISA測(cè)定兔抗血清效價(jià)為64 000，稀釋500倍使用。Western-Blot檢測(cè)CD3重組蛋白與兔多抗的結(jié)合。

表1 引物列表

1.4 單克隆抗體

鼠抗牙鲆紅細(xì)胞單抗[22]和鼠抗牙鲆IgM單抗[19]由實(shí)驗(yàn)室前期制備，本論文中應(yīng)用2株單抗的腹水，效價(jià)200 000，使用時(shí)按1∶1 000稀釋。

1.5 遲緩愛德華氏菌

遲緩愛德華氏菌由本實(shí)驗(yàn)室保種培養(yǎng)，將保種的遲緩愛德華氏菌接種到滅菌的BHI液體培養(yǎng)基中，28 ℃培養(yǎng)24 h后，離心，收集菌體，一部分用無菌PBS重懸，以比濁儀測(cè)定濃度，PBS調(diào)整至以1×107CFU/mL，用于感染實(shí)驗(yàn)；另一部分菌體以4%福爾馬林(V/V)重懸，28 ℃滅活48 h后，離心，收集滅活菌體，PBS重懸，接種到BHI固體培養(yǎng)基培養(yǎng)24 h，無菌體生長，則以比濁儀測(cè)定濃度，PBS調(diào)整至1×107CFU/mL為滅活疫苗，用于免疫實(shí)驗(yàn)。

1.6 感染和免疫實(shí)驗(yàn)

在魚類細(xì)菌感染或疫苗免疫相關(guān)研究中，通常采用腹腔注射每20 g魚體重1 mL 1×107CFU/mL菌體或疫苗，由于本文中選用的遲緩愛德華氏菌是一種對(duì)牙鲆致病性較強(qiáng)的病原菌，為不使實(shí)驗(yàn)過程中出現(xiàn)注射過量病原引起應(yīng)激死亡等情況，并結(jié)合前期預(yù)實(shí)驗(yàn)，故選擇每尾牙鲆注射200 μL的活菌液或滅活疫苗。將750尾牙鲆隨機(jī)分為3組，每組250尾，感染組每尾注射遲緩愛德華氏菌液200 μL，免疫組每尾注射200 μL滅活疫苗，對(duì)照組注射200 μL PBS。每組分別于注射后第0、1、3、5、7、9、14、21和28天隨機(jī)選取20尾牙鲆抽取外周血、提取白細(xì)胞，用于流式細(xì)胞術(shù)檢測(cè)。

1.7 免疫雙熒光和流式細(xì)胞術(shù)

將提取的白細(xì)胞樣品300 μL加入離心管，20%牛血清白蛋白封閉2 h，PBS洗3次，每次5 min；加入等體積比例混合的鼠抗牙鲆IgM單抗和兔抗牙鲆CD3多抗為第一抗體，37 ℃孵育1 h，PBS洗3次，每次5 min；再以FITC標(biāo)記的羊抗鼠IgG(購于美國Sigma公司)和Cy5標(biāo)記的羊抗兔IgG(購于博奧森生物技術(shù)公司)的混合抗體為第二抗體，37 ℃孵育30 min，PBS洗3次，每次5 min。此時(shí)用移液槍吸取白細(xì)胞懸液20 μL滴于載玻片上，避光沉降30 min，棄去上清；加4%多聚甲醛固定10 min，PBS洗3次，每次5 min；加20 μL DAPI染液(4,6-聯(lián)脒-2-苯基吲哚，細(xì)胞核染色劑)，室溫靜置15 min；PBS洗3次，每次5 min；室溫干燥，甘油封片，于熒光顯微鏡(Olympus，日本)下觀察T、B淋巴細(xì)胞并拍照；余下部分用于上樣流式細(xì)胞儀檢測(cè)。上樣時(shí)對(duì)照有3組：①以PBS代替一抗，標(biāo)記FITC和Cy5的混合抗體為二抗；②以鼠抗牙鲆IgM單抗為一抗，以FITC標(biāo)記的羊抗鼠IgG為二抗；③兔抗牙鲆CD3多抗為一抗，Cy5標(biāo)記的羊抗兔IgG為二抗。本實(shí)驗(yàn)重復(fù)3次。上樣流式時(shí)先通過前向角(FSC)/側(cè)向角(SSC)的散點(diǎn)圖射門細(xì)胞群，然后通過FL1/SSC和FL1/FL4檢測(cè)熒光陽性細(xì)胞群(FL1為綠色熒光通道，F(xiàn)L4為紅色熒光通道)。

測(cè)定外周血細(xì)胞時(shí)，以300 μL稀釋600倍的外周血細(xì)胞為抗原，鼠抗牙鲆紅細(xì)胞單抗為一抗，F(xiàn)ITC標(biāo)記的羊抗鼠IgG為二抗，對(duì)照組以PBS代替一抗。

1.8 吉姆薩染色

取上樣流式細(xì)胞術(shù)的牙鲆外周血細(xì)胞20 μL和提取的白細(xì)胞20 μL滴于載玻片上，沉降30 min，棄去上清，加4%多聚甲醛固定10 min，PBS洗3次，每次5 min，進(jìn)行吉姆薩染色，顯微鏡下觀察血細(xì)胞形態(tài)并拍照。

1.9 數(shù)據(jù)分析

實(shí)驗(yàn)所得數(shù)據(jù)均以平均值±標(biāo)準(zhǔn)差表示，用SPSS 19.0統(tǒng)計(jì)軟件進(jìn)行方差分析和Duncan多重比較，當(dāng)P<0.01時(shí)差異顯著，利用Origin 8.5進(jìn)行制圖。

2 結(jié)果

2.1 牙鲆CD3分子蛋白的重組表達(dá)和兔多抗的制備

經(jīng)誘導(dǎo)的重組CD3大腸桿菌的全菌蛋白中出現(xiàn)了一條相對(duì)分子量約為27 kDa的蛋白條帶，符合預(yù)期結(jié)果；未誘導(dǎo)的重組菌中沒有出現(xiàn)相對(duì)應(yīng)大小的條帶；純化后蛋白條帶單一(見圖1 A)。Western-Blot結(jié)果顯示，在27 kDa處有單一蛋白條帶顯色(見圖1 B)。結(jié)果表明，制得的多抗能夠與重組CD3蛋白特異性結(jié)合。

(圖A中：M，標(biāo)準(zhǔn)分子量蛋白；1，未誘導(dǎo)重組菌體總蛋白；2，誘導(dǎo)后重組菌體總蛋白；3，純化后的重組蛋白。圖B中：M，標(biāo)準(zhǔn)分子量蛋白；1，純化的CD3重組蛋白。Fig.A: M： Marker; 1： Total protein from uninduced BL21 containing recombinant protein; 2： Total proteins from induced BL21containing recombinant protein; 3： Purified CD3 recombinant protein. Fig.B: M： Marker; 1： Purified CD3 recombinant protein.)

圖1 牙鲆CD3蛋白重組表達(dá)的SDS-PAGE(A)及CD3重組蛋白與兔多抗的Western-Blot(B)

Fig.1 SDS-PAGE analysis of recombinant CD3 protein expression(A) and CD3 recombinant protein with rabbit anti-CD3 polyclonal antibodies in Western-Blot (B)

2.2 免疫雙熒光

熒光顯微鏡下，一部分白細(xì)胞呈現(xiàn)明顯的綠色熒光為FITC標(biāo)記的IgM+細(xì)胞，同一視野下還有一部分白細(xì)胞呈現(xiàn)出明顯的紅色熒光為Cy5標(biāo)記的CD3+細(xì)胞，并且IgM+細(xì)胞與CD3+細(xì)胞之間無交叉；沒有熒光信號(hào)的細(xì)胞為IgM-/CD3-白細(xì)胞(見圖2)。結(jié)果表明，鼠抗IgM單抗和兔抗CD3多抗分別能夠特異性識(shí)別B淋巴細(xì)胞和T淋巴細(xì)胞，并且無交叉反應(yīng)。

(1：熒光顯微鏡下鼠抗IgM單抗與牙鲆白細(xì)胞間接免疫熒光結(jié)果；2：熒光顯微鏡同一視野下兔抗CD3多抗與牙鲆白細(xì)胞間接免疫熒光結(jié)果；3：熒光顯微鏡同一視野下牙鲆白細(xì)胞DAPI核染色結(jié)果；4:圖1、2和3的合成圖；標(biāo)尺=10 μm。1: The indirect immunofluorescence double-staining result of mouse anti-IgM mab specifically reacted with the leukocytes ofParalichthysolivaceusunder the fluorescence microscope; 2: The indirect immunofluorescence double-staining result of rabbit anti-CD3 recombinant protein polyclonal antibodies specifically reacted with the peripheral leukocytes ofParalichthysolivaceusunder the fluorescence microscope in the same vision; 3: Leukocytes ofParalichthysolivaceusnuclear staining with DAPI under the fluorescence microscope in the same vision; 4: Composite image of image 1, 2 and 3.(bar=10 μm).)

圖2 鼠抗IgM單抗和兔抗CD3多抗與牙鲆白細(xì)胞的免疫雙熒光染色

Fig.2 Mouse anti-IgM mab and rabbit anti-CD3 recombinant protein polyclonal antibodies specifically reacted with the leukocytes of Paralichthys olivaceus by indirect immunofluorescence double-staining

2.3 外周血細(xì)胞吉姆薩染色結(jié)果

吉姆薩染色顯示，牙鲆外周血中多為紅細(xì)胞，有少量白細(xì)胞。牙鲆紅細(xì)胞大小為8～9 μm，呈橢圓形，核圓形，胞核染色呈深藍(lán)色，胞質(zhì)無顆粒，染成紅色(見圖3-1)。提取的白細(xì)胞大部分為淋巴細(xì)胞，其次是血栓細(xì)胞，還有少量單核細(xì)胞，粒細(xì)胞較少。淋巴細(xì)胞大小為3～4 μm，呈圓形或不規(guī)則圓形，胞核呈橢圓或馬蹄形，位于中央，核染深藍(lán)色，胞質(zhì)淡藍(lán)紫色；血栓細(xì)胞大小為5～6 μm，呈紡錘形或淚滴狀，胞核大，卵圓形位于中央，核染深藍(lán)色，胞質(zhì)淺紫色；單核細(xì)胞大小為6～7 μm，呈球形，胞核呈腎形或馬蹄形，位于一側(cè)或中央，核染深藍(lán)色，胞質(zhì)藍(lán)紫色或淡紫色(見圖3-2)。

(1：外周血全血細(xì)胞；2：提取的白細(xì)胞；標(biāo)尺=10 μm。1: The PBL cells ; 2: The leukocytes.(bar=10 μm).)

圖3 牙鲆外周血全血細(xì)胞和白細(xì)胞吉姆薩染色結(jié)果

Fig.3 the peripheral blood ( PBL) cells and the leukocytes ofParalichthysolivaceusstained with Gimesa

2.4 感染和免疫后牙鲆外周血細(xì)胞濃度的變化

牙鲆紅細(xì)胞單抗的流式細(xì)胞術(shù)，F(xiàn)SC/SSC的流式散點(diǎn)圖顯示，牙鲆外周血細(xì)胞為一群細(xì)胞(見圖4 A)；FL1/SSC的流式散點(diǎn)圖結(jié)果顯示，外周血細(xì)胞分為2群，綠色的FITC標(biāo)記陽性一群為紅細(xì)胞比例為(90±1.9)%，另一群陰性細(xì)胞為白細(xì)胞比例占(9.2±1.2)%(見圖4 B)。按照抽取血液體積稀釋比例計(jì)算，對(duì)照組，牙鲆外周血中紅細(xì)胞濃度為(2.8±0.62)×109cells/mL；遲緩愛德華氏菌感染和免疫后，紅細(xì)胞比例(90±2.6)%和濃度(2.9±0.45)×109cells/mL，與對(duì)照組差異不顯著。感染和免疫后，白細(xì)胞濃度均顯著高于對(duì)照組。感染組，外周血中白細(xì)胞濃度于第3天開始顯著上升，此時(shí)白細(xì)胞濃度為(1.9±1.5)×108cells/mL，對(duì)照組為(6.9±2.2)×107cells/mL；直到第14天達(dá)到最大值，此時(shí)白細(xì)胞濃度為(4.7±1.7)×108cells/mL，對(duì)照組為(6.9±1.6)×107cells/mL；隨后開始下降，第28天時(shí)白細(xì)胞濃度降至對(duì)照組水平為(5.0±1.7)×107cells/mL。免疫組，外周血中白細(xì)胞濃度同樣于第3天開始顯著上升，此時(shí)白細(xì)胞濃度為(1.9±0.6)×108cells/mL，

(A：牙鲆外周血細(xì)胞散點(diǎn)圖；B：紅細(xì)胞熒光散點(diǎn)圖。A：FSC/SSC dot plot in FCM；B：FL1/SSC scatter plot of red cells fluorescent in FCM with gated cells.)

圖4 應(yīng)用紅細(xì)胞單抗檢測(cè)牙鲆外周血細(xì)胞的流式細(xì)胞術(shù)結(jié)果

Fig.4 Flow cytometry analysis of the PBL cells by using Mab against red blood cells

(圖中不同字母表示同一時(shí)間點(diǎn)不同處理間差異顯著,P<0.01。Different letters meant significant differences at 0.01 levels among treatments at the same time point，P<0.01.)

圖5 感染和免疫后牙鲆外周血中白細(xì)胞總濃度的變化結(jié)果

Fig.5 Variation of concentration of leukocytes in PBL fromParalichthysolivaceusafter infected or immunized

對(duì)照組為(6.9±2.2)×107cells/mL；第14天達(dá)到最大值，此時(shí)白細(xì)胞濃度為(7.1±1.8)×108cells/mL，對(duì)照組為(6.9±1.6)×107cells/mL；第28天降至(1.6±1.7)×108cells/mL，對(duì)照組為(5.0±1.7)×107cells/mL(見圖5)。

2.5 感染和免疫后牙鲆外周血T、B淋巴細(xì)胞數(shù)量的變化

牙鲆外周血白細(xì)胞流式散點(diǎn)圖顯示，紅色部分表示Cy5標(biāo)記陽性的T細(xì)胞比例，綠色部分表示FITC標(biāo)記陽性的IgM+B細(xì)胞比例，兩者無交叉(見圖6)。感染組的T淋巴細(xì)胞比例第1天就開始顯著上升，此時(shí)T淋巴細(xì)胞比例為(9.8±0.8)%，對(duì)照組為(8.2±0.9)%；第9天達(dá)到最大值，此時(shí)T淋巴細(xì)胞比例為(24.3±1.28)%，對(duì)照組為(8.5±1.38)%；隨后逐漸降低，至第28天時(shí)，感染組降至(12.8±1.3)%，對(duì)照組為(7.7±1.68)%。免疫組T淋巴細(xì)胞比例從第1天也有上升，但第5天才開始顯著上升，此時(shí)T淋巴細(xì)胞比例為(12.1±1.5)%，對(duì)照組為(7.4±1.6)%；第9天達(dá)到最大值，此時(shí)T淋巴細(xì)胞比例為(20.5±1.12)%，對(duì)照組為(8.5±1.38)%；隨后逐漸降低，至第28天時(shí)，免疫組降至(10.9±0.5)%，對(duì)照組為(7.7±1.68)%(見圖7)。

感染組IgM+B淋巴細(xì)胞比例自第1天起顯著高于對(duì)照組，此時(shí)IgM+B細(xì)胞比例為(19.3±1.63)%，對(duì)照組為(14.3±1.05)%；于第21天達(dá)到最大值，此時(shí)IgM+B細(xì)胞比例為(40.9±1.52)%，對(duì)照組為(13.77±1.56)%；在第28天時(shí)IgM+B細(xì)胞比例有所降低，為(32.1±1.26)%，對(duì)照組為(16.02±1.79)%。免疫組IgM+B細(xì)胞比例也從第1天開始顯著升高，此時(shí)IgM+B細(xì)胞比例為(18.6±0.86)%，對(duì)照組為(14.3±1.05)%；于第21天達(dá)到最大值，此時(shí)IgM+B細(xì)胞比例為(39.3±1.55)%，對(duì)照組為(13.77±1.56)%；第28天時(shí)降低為(30.9±1.62)%，對(duì)照組為(16.02±1.79)%(見圖8)。

(A：FSC/SSC散點(diǎn)圖；B：FL1/FL4雙熒光散點(diǎn)圖。A：FSC/SSC dot plot in FACS；B：FL1/FL4 scatter plot of double fluorescent in FACS with gated cells.)

圖6 應(yīng)用IgM單抗和CD3多抗檢測(cè)牙鲆外周血白細(xì)胞的流式細(xì)胞術(shù)結(jié)果

Fig.6 Flow cytometry analysis of the leukocytes by using Mab against IgM and Pab against CD3

(圖中不同字母表示同一時(shí)間點(diǎn)不同處理間差異顯著,P<0.01。Different letters meant significant differences at 0.01 levels among treatments at the same time point，P<0.01.)

圖7 感染和免疫后牙鲆外周血白細(xì)胞中T細(xì)胞比例變化結(jié)果

Fig.7 Variation of T cells percentage in PBL leukocytes in FACS after infected or immunized

(圖中不同字母表示同一時(shí)間點(diǎn)不同處理間差異顯著,P<0.01。Different letters meant significant differences at 0.01 levels among treatments at the same time point，P<0.01.)

圖8 感染和免疫后牙鲆外周血白細(xì)胞中IgM+B細(xì)胞比例變化結(jié)果

Fig.8 Variation of IgM+B cells percentage in PBL leukocytes in FACS after infected or immunized

結(jié)果表明，感染和免疫后的牙鲆T、B淋巴細(xì)胞比例均比對(duì)照組顯著升高，并且感染組在免疫應(yīng)答過程中外周血T、B淋巴細(xì)胞比例變化趨勢(shì)顯著高于免疫組，T細(xì)胞和IgM+B細(xì)胞的數(shù)量與病原感染或刺激顯著相關(guān)。

3 討論

本文根據(jù)已發(fā)表的牙鲆T細(xì)胞表面標(biāo)志分子CD3的基因序列，重組表達(dá)出CD3蛋白，并制備了兔抗CD3多抗。應(yīng)用兔抗CD3多抗和鼠抗IgM單抗的免疫雙熒光技術(shù)，熒光顯微鏡下觀察到CD3+T細(xì)胞和IgM+B細(xì)胞之間無交叉，說明CD3多抗和IgM單抗能夠特異性地識(shí)別牙鲆的T、B淋巴細(xì)胞。

由于牙鲆的外周血細(xì)胞在流式細(xì)胞儀中不能根據(jù)細(xì)胞大小和顆粒度(即FSC/SSC)分為不同的細(xì)胞群，所以論文選擇應(yīng)用牙鲆的紅細(xì)胞單抗的流式細(xì)胞術(shù)來檢測(cè)外周血細(xì)胞。本文應(yīng)用牙鲆紅細(xì)胞單抗、IgM單抗和兔抗CD3多抗的流式細(xì)胞術(shù)研究了牙鲆在感染和免疫遲緩愛德華氏菌后外周血細(xì)胞及T、B淋巴細(xì)胞的的數(shù)量變化。結(jié)果發(fā)現(xiàn)，感染和免疫后，牙鲆外周血紅細(xì)胞數(shù)量無顯著變化，而白細(xì)胞的數(shù)量在魚體分別受到2種刺激后均呈現(xiàn)顯著升高的趨勢(shì)，在14天后又逐漸降低至接近正常水平。這與K?llner[23]以嗜水氣單胞菌滅活疫苗注射虹鱒(Oncorhynchusmykiss)和單紅[24]給南方鲇注射嗜水氣單胞菌滅活疫苗后，外周血的白細(xì)胞數(shù)量顯著升高的研究結(jié)果相似。這是因?yàn)樵谑艿讲【蛞呙绱碳さ难装Y初期，非特異性免疫的防御使得各類具有吞噬作用的白細(xì)胞大量增殖[23]，包括單核細(xì)胞、中性粒細(xì)胞，以抵御病菌感染，增強(qiáng)魚體抗病力，隨后魚體逐漸以特異性免疫為主，具有吞噬作用的白細(xì)胞數(shù)量減少，T、B淋巴細(xì)胞增殖導(dǎo)致比例上升。本論文的實(shí)驗(yàn)結(jié)果也證明了這一點(diǎn)，牙鲆在感染和免疫后T、B淋巴細(xì)胞比例均顯著升高，直到第9天T細(xì)胞比例達(dá)到最大值，這與雛雞感染艾美爾球蟲后外周血T細(xì)胞比例在第7天達(dá)到最大值相似[26]。在感染和免疫后的第21天，IgM+B淋巴細(xì)胞比例達(dá)到最大值，是因?yàn)樵诓≡秩脒^程中，魚體要通過淋巴細(xì)胞的增殖和免疫球蛋白的增多，來抵抗病原菌的刺激[23-25]。T、B淋巴細(xì)胞比例的變化過程中，感染組均顯著高于免疫組，這可能是由于活菌被注射入魚體內(nèi)后，在適宜的溫度下迅速增殖，給予機(jī)體持續(xù)刺激，使魚體的免疫應(yīng)答比免疫組更強(qiáng)。

有關(guān)魚類免疫恢復(fù)期的研究目前根據(jù)魚種類的不同有差異，有研究表明，全菌滅活疫苗免疫中華倒刺鲃(SpinibarbussinensisBleeker)后，外周血中白細(xì)胞數(shù)量第4～7天達(dá)到峰值，于第28天降低至對(duì)照水平；外周血中淋巴細(xì)胞的百分比于第21天到達(dá)峰值隨后下降，于第35天下降至對(duì)照水平[25]；南方鲇(Silurusmeridionalis)[24]、斑點(diǎn)叉尾鮰(Ictaluruspunctatus)[34]等魚類的免疫相關(guān)實(shí)驗(yàn)中也得到類似結(jié)果，魚類在28天左右達(dá)到對(duì)照水平；牙鲆浸泡免疫的愛德華氏菌滅活疫苗6周內(nèi)，其外周血白細(xì)胞中IgM+B細(xì)胞比例亦出現(xiàn)先升高后降低[35]，并且浸泡疫苗濃度低時(shí)間短的先在第4周達(dá)到峰值，浸泡濃度高時(shí)間長的在第5周達(dá)到峰值。本文實(shí)驗(yàn)中牙鲆在感染或免疫后，外周血中白細(xì)胞數(shù)量、T、B淋巴細(xì)胞的比例于第28天時(shí)雖有降低，仍顯著高于對(duì)照水平。這可能與免疫或感染時(shí)菌的濃度有關(guān)，或者是牙鲆本身免疫恢復(fù)期稍長，后續(xù)實(shí)驗(yàn)會(huì)延長至其恢復(fù)對(duì)照水平為止。

在高等動(dòng)物中，根據(jù)各種類型淋巴細(xì)胞的數(shù)量變化范圍能夠判斷其處于感染或健康狀態(tài)[33]。而在魚類作為變溫動(dòng)物，水溫、鹽度、pH值等均是影響魚類生理指標(biāo)的關(guān)鍵因素，研究表明，在注射嗜水氣單胞菌滅活疫苗(實(shí)驗(yàn)水溫為13 ℃左右)后第2天，南方鲇外周血白細(xì)胞數(shù)量在第4～28天有顯著上升[24]。在注射愛德華氏菌滅活疫苗(實(shí)驗(yàn)水溫為25 ℃左右)后第2天，斑點(diǎn)叉尾鮰外周血白細(xì)胞數(shù)量在第4～14天有顯著上升[34]。結(jié)合本文實(shí)驗(yàn)結(jié)果，在感染或免疫后(溫度為20～23 ℃)第3～28天時(shí)，牙鲆外周血白細(xì)胞、T、B淋巴細(xì)胞的數(shù)量或比例均已顯著高于對(duì)照組。并且在研究牙鲆外周血白細(xì)胞數(shù)量時(shí)，觀察到血栓細(xì)胞的數(shù)量也有著較為明顯的變化，可能與牙鲆的免疫應(yīng)答也有著重要關(guān)系。有報(bào)道利用血栓細(xì)胞單抗分離出鯉魚的血栓細(xì)胞[27]，并做了一系列相關(guān)研究，但血栓細(xì)胞的數(shù)量是否能作為評(píng)估指標(biāo)還需要進(jìn)一步研究。要探究魚類淋巴細(xì)胞與生理狀態(tài)的關(guān)系還需要掌握魚體與環(huán)境因子、病原感染等相關(guān)性的詳細(xì)數(shù)據(jù)來確定。

在魚類機(jī)體受刺激后外周血T細(xì)胞數(shù)量的研究還不全面，大多數(shù)研究以基因水平變化為主。研究表明抗B淋巴細(xì)胞的有效抗體較多，抗魚類T淋巴細(xì)胞的有效抗體相對(duì)較少，這可能跟T細(xì)胞表面缺少可結(jié)合的受體有關(guān)[28]。然而，隨著T淋巴細(xì)胞表面標(biāo)志的發(fā)現(xiàn)，抗表面標(biāo)志物的抗體開始被應(yīng)用于細(xì)胞類群的鑒別，如CD4+T細(xì)胞和CD8+T細(xì)胞的分類研究也越來越多[21,29-30]。研究表明，T細(xì)胞表面的CD4和CD8分子不能同時(shí)表達(dá)于同一細(xì)胞，識(shí)別外源性抗原的細(xì)胞為CD4+的Th細(xì)胞，識(shí)別內(nèi)源性抗原的細(xì)胞為CD8+的細(xì)胞毒性T細(xì)胞[21,29-30]。臨床上，CD4+T細(xì)胞與CD8+T細(xì)胞的比值是評(píng)估機(jī)體健康的重要指標(biāo)，如北京地區(qū)健康成人CD3+細(xì)胞陽性率50.7%～86.0%，CD4+細(xì)胞陽性率23.3%～50.2%，CD8+細(xì)胞陽性率12.5%～36.9%,CD4/CD8比值為0.80～2.74[31]。牙鲆的T細(xì)胞表面標(biāo)志CD4、CD8已經(jīng)被克隆表達(dá)出來，開始應(yīng)用于牙鲆免疫相關(guān)的研究[32]。

4 結(jié)語

本研究制備了牙鲆T淋巴細(xì)胞表面標(biāo)志物CD3的多克隆抗體，主要采用流式細(xì)胞術(shù)對(duì)愛德華氏菌感染和滅活疫苗免疫后牙鲆外周血細(xì)胞的數(shù)量進(jìn)行了檢測(cè)。此研究方法較常規(guī)染色計(jì)數(shù)細(xì)胞的方法更為特異和準(zhǔn)確，在魚類血細(xì)胞數(shù)量尤其是牙鲆淋巴細(xì)胞數(shù)量的研究上有重要意義。研究結(jié)果顯示，在感染和免疫后牙鲆外周血白細(xì)胞數(shù)量、T、B淋巴細(xì)胞的比例均呈現(xiàn)先顯著上升后逐漸降低的趨勢(shì)，并且感染組T、B淋巴細(xì)胞比例顯著高于免疫組。為進(jìn)一步地探究牙鲆T淋巴細(xì)胞亞群可否作為評(píng)估指標(biāo)，本實(shí)驗(yàn)室已經(jīng)著手做了關(guān)于牙鲆T淋巴細(xì)胞亞群的表面分子CD4、CD8的相關(guān)工作，以期為牙鲆健康評(píng)估體系提供更全面的數(shù)據(jù)參考。

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責(zé)任編輯 朱寶象

Numerical Change of Peripheral Blood Cells of Flounder (Paralichthys olivaceus) After Edwardsiellatarda Infection and Vaccination

ZHEN Meng-Xiao, XING Jing, SHENG Xiu-Zhen, TANG Xiao-Qian, ZHAN Wen-Bin

(Laboratory of Pathology and Immunology of Aquatic Animals, Ocean University of China, Qingdao 266003, China)

Fish blood cells involve in the process of immune response and anti-infection. The leukocytes, especially T and B lymphocytes, play important roles in fish immunity, and in the meantime, the number of blood cells or lymphocytes vary during the physiologic processes. In this study, the variation of peripheral blood cells of flounder (Paralichthysolivaceus) afterEdwardsiellatardainfection and vaccination was determined. Our primary research has produced anti-erythrocytes monoclonal antibody (Mab) and anti-immunoglobulin (IgM) Mab, respectively. In this study, the CD3 molecule was cloned and its recombined protein was expressed, and then rabbit anti-CD3 polyclonal antibody (RaPoCD3-Pab) was produced. T and B lymphocytes in peripheral blood (PBL) cells were observed and counted by flow cytometry (FCM) using RaPoCD3-Pab and IgM Mab, respetively. Flounder was intraperitoneally injected with 1×107CFU/mLEdwardsiellatarda, its inactivated vaccine and PBS, respectively. The PBL was collected on days 0, 1, 3, 5, 7, 9, 14, 21 and 28 post injection, respectively. Erythrocytes and leukocytes were analyzed by double fluorescent FCM using Mab against erythrocytes of flounder, and T and B lymphocytes were counted by double fluorescent FCM with RaPoCD3-Pab and IgM Mab. The result showed that the RaPoCD3-Pab and IgM Mab specifically recognized T and B lymphocytes, which were labeled by red or green fluorescence, respectively, and no cross reaction was detected. The T cells were in percentage of (7.49±1.5)% and B cells were (14.64±1.8)% in leukocytes. After infection or vaccination, the concentration of erythrocytes had no significant variation (P<0.01, ANOVA) during the whole experiment compared with the control group, keeping at (2.9±0.45)×109cells/mL in PBL. The leukocyte concentration in control group maintained at (6.9±1.6)×107cells/mL, the proportion of T cells was (8.05±1.38)%, the proportion of B cells was (13.77±1.56)%. In the infection group, the concentration of leukocyte increased significantly on the 3rdday, then reached the peak at (4.7±1.7)×108cells/mL on the 14thday. The proportion of T cells rose immediately on the 1stday, then reached the maximum of (24.3±1.28)% on the 9thday. The proportion of B cells increased on the 1stday, and reached the peak (40.9±1.52) % on the 21stday (P<0.01, ANOVA). In the vaccination group, the leukocyte concentration increased significantly on the 3rdday, then reached the peak of (7.1±1.8)×108cells/mL on the 14thday. The proportion of T cells rose significantly since the 5thday, then reached the maximum of (20.5±1.12)% on the 9thday. The proportion of B cells increased on the 1stday, and reached the peak of (39.3±1.55)% on the 21stday (P<0.01, ANOVA). The results indicated that leukocyte concentration, the proportion of T and B cells in PBL increased significantly before the 21thday after infection and vaccination, leukocyte concentration kept rising during a period of 3-14 days, T cells percentage rose till the 9thday, B cells percentage climbed up till the 21thday, the proportions of T and B cells in PBL increased significantly after infection and vaccination, especially on the 1stday. The counts of leukocytes or the percentage of T and B cells varied with the infection or vaccination process, which might be potential indexes for fish physiologic processes.

Paralichthysolivaceus; infection; vaccination;Edwardsiellatarda; T lymphocyte; B lymphocyte

國家重點(diǎn)基礎(chǔ)研究發(fā)展計(jì)劃項(xiàng)目(2012CB114406)；國家自然科學(xué)基金項(xiàng)目(31172429，31302216)資助 Support by National Program on Key Basic Research Project (2012CB114406); National Natural Science Foundation of China (31172429，31302216)

2016-03-02；

2016-05-06

甄夢(mèng)曉(1989-)，女，碩士生，主要從事水產(chǎn)動(dòng)物病害與免疫學(xué)研究。E-mail：zhenxiah2009@163.com

** 通訊作者：E-mail：xingjing@ouc.edu.cn

S941.4；S91

A

1672-5174(2017)03-034-09

10.16441/j.cnki.hdxb.20160053

甄夢(mèng)曉， 邢婧， 繩秀珍， 等. 遲緩愛德華氏菌感染和疫苗免疫后牙鲆的血細(xì)胞數(shù)量變化[J].中國海洋大學(xué)學(xué)報(bào)(自然科學(xué)版),2017, 47(3): 34-42.

ZHEN Meng-Xiao, XING Jing, SHENG Xiu-Zhen, et al. Numerical change of peripheral blood cells of flounder (Paralichthysolivaceus) afterEdwardsiellatardainfection and vaccination [J].Periodical of Ocean University of China, 2017, 47(3): 34-42.