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1 杂色鲍血细胞分类 结构和免疫功能的流式细胞术分析 1, 冼健安 2, 钱坤 2, 郭慧 3, 王冬梅 1, 张秀霞 1, 苗玉涛 2, 潘训彬 2 2, 王安利 (1., ; 2.,, ; 3.,, ) 摘要 : 应用流式细胞术对杂色鲍 (Haliotis diversicolor Reeve) 血细胞的分类 结构和免疫功能进行分析 根据细胞前向角散射光 (FSC) 和侧向角散射光 (SSC) 强度的不同, 可将血细胞分为三个亚群 : 透明细胞 小颗粒细胞和大颗粒细胞, 组成比例分别为 32.71% 58.17% 和 8.55% 血细胞的平均总凋亡和死亡率为 3.76% 血细胞对荧光微球的总吞噬率为 63.67%, 其中吞噬 1 个 2 个 3 个及以上荧光微球的血细胞分别占 22.31% 16.39% 24.96% 线粒体数量 溶酶体数量 非特异性酯酶活性和非诱导性活性氧 (ROS) 含量均在大颗粒细胞中最高, 透明细胞最低 结果表明, 杂色鲍三类血细胞在结构和功能上均存在差异, 两类颗粒细胞可能在鲍类免疫过程中发挥着更为重要的作用 关键词 : 杂色鲍 ; 流式细胞术 ; 血细胞 ; 免疫中图分类号 : S917.4; Q952 文献标识码 : A 文章编号 : (2015) doi: /hykx (Haliotis diversicolor),,,,, [1], [2-5],, (Flow Cytometry, FCM) 20 70,, [6-8] [9-12], [13-14] FCM,, FCM 1 材料和方法 1.1 材料与试剂 (Haliotis diversicolor Reeve), 6~7cm, ( 30, 20~22 ) 1,, (FDA) 2, 7 - (DCFH-DA) Sigma, Annexin V- FITC/PI( ) LysoTracker Red MitoTracker Green (yellow-green fluorescent carboxylate-modified FluoSpheres beads, 1 µm) Invitrogen, 1.2 血细胞悬液的制备, : ; : : (S ); (ITBB2015ZY01 ITBB2015ZD03) : (1983-),,,, :,, : , xian-ja@163.com; (1957-),,,,, :, : , wanganl@scnu.edu.cn. 8 / 2015 / 39 / 12

2 1.. 1, 10 6 /ml 10, 1.3 流式细胞仪 BD(Becton Dickinson) FACSCalibur, CellQuest (Forward light scatter, FSC), (Side light scatter, SSC) ; FITC( ) MitoTracker Green FDA DCF(2, 7 - ) (FL1), PI LysoTracker Red (FL2) 1.4 血细胞分类与组成比例 200, SSC FSC, SSC-FSC, 1.5 血细胞凋亡率 Annexin V-FITC/PI, 1x Annexin V, /ml, 100 µl 5 µl Annexin V-FITC 10 µl PI, 15 min, 400 µl 1x Annexin V, 200, Annexin V-FITC (FL1), PI (FL2) 2, : (Annexin V-FITC-/PI-) a, (Annexin V-FITC+/PI-) b, (Annexin V-FITC+/PI+) c, ( b+c), 30 min 1.6 吞噬活性 400 µl, 10 µl 1/10, 1 h, 200, FL1,, 1.7 线粒体数量 MitoTracker Green, MitoTracker Green 200 µl, 50 nmol/l MitoTracker Green 30 min, 200, MitoTracker Green (FL1), SSC- FSC, MitoTracker Green, MitoTracker Green, Mito- Tracker Green 1.8 溶酶体数量 LysoTracker Red, DND99,, 200 µl, 50 nmol/l LysoTracker Red 60 min, 200, LysoTracker Red (FL2), SSC-FSC, LysoTracker Red, LysoTracker Red, LysoTracker Red 1.9 非特异性酯酶活性 FDA 200 µl, 5 µmol/lfda 30 min, 200, FDA (FL1), SSC-FSC, FDA, FDA, FDA Marine Sciences / Vol. 39, No. 12 /

3 1.10 活性氧 (ROS) 含量 DCFH-DA ROS 200 µl, 10 µmol/ldcfh-da 30 min, 200, DCF (FL1), SSC-FSC, DCF, DCF, DCF ROS 1.11 统计分析 ± (Mean ± SD), SPSS 13.0 Tukey, P< 结果 2.1 血细胞分类与组成比例 1A, 1B,,, 1, FSC SSC, : R1, R2 R3 R1 FSC SSC,,, ; R3 FSC SSC,,, R2 R1 R3, FSC SSC 1,, 1, A (R3), B ; 2, : > >, (P<0.05) 2.2 细胞凋亡率 2 Annexin V-FITC/PI Fig.1 1 SSC-FSC (A) (B) SSC-FSC dot plot (A) and contour plot (B) of H. diversicolor hemocytes R1. ; R2. ; R3. R1. Hyalinocyte; R2. Small granulocyte; R3. Large granulocyte 表 1 杂色鲍 3 类血细胞的大小和颗粒复杂度 (A.U.) Tab.1 Size and granular complexity of three hemocyte subpopulations from H. diversicolor (A.U.) (FSC ) (SSC ) (R1) 77.98±2.00 c ±4.35 c (R2) ±11.15 b ±9.81 b (R3) ±11.95 a ±42.46 a : (P<0.05), n= / 2015 / 39 / 12

4 表 2 杂色鲍 3 类血细胞的组成比例 (%) Tab.2 Proportion of three hemocyte subpopulations from H. diversicolor (%) (R1) 32.71±7.18 b (R2) 58.17±5.53 a (R3) 8.55±2.68 c ( M2), 3 ( M3), M4 1 4, %; %,, 8.17%, 21.76%; %; 63.67% 2 Annexin V-FITC/PI Fig.2 Apoptosis dot plot of H. diversicolor hemocytes stained with Annexin V-FITC and PI a(annexin V-FITC-/PI-). ; b(annexin V-FITC+/ PI-). ; c (Annexin V-FITC+/PI+). Quadrant a (Annexin V-FITC-/PI-) live cells; quadrant b (Annexin V-FITC+/PI-) early apoptotic cells; quadrant c (Annexin V-FITC+/ PI+) late apoptotic and necrotic cells 10, 3, 3.76% 表 3 杂色鲍血细胞凋亡率 (%) Tab.3 Apoptotic ratio of H. diversicolor hemocytes (%) ( b) 1.27± ( c) 2.49± ( b+c) 3.76± : n= 吞噬活性 3, 1 2 3, 1 ( M1), 2 3 Fig.3 Distribution histogram of H. diversicolor hemocytes phagocytosed fluorescent beads M1. 1 ; M2. 2 ; M3. 3 ; M4. 1 M1, region of one hemocyte phagocytosed fluorescent bead; M2, region of two hemocytes phagocytosed fluorescent beads; M3, region of three or more hemocytes phagocytosed fluorescent beads; M4, region of one or more hemocytes phagocytosed fluorescent beads 表 4 杂色鲍血细胞的吞噬活性 (%) Tab.4 Phagocytic activity of H. diversicolor hemocytes (%) 1 (M1) 22.31± (M2) 16.39± (M3) 24.96± (M4) 63.67± : n= 线粒体数量和溶酶体数量 5,,, 3.4, 10.4 ; 3.7, 10.2 (P<0.05) Marine Sciences / Vol. 39, No. 12 /

5 表 5 杂色鲍 3 类血细胞的生理特征 Tab.5 Physiological characteristics of three hemocyte subpopulations from H. diversicolor (A.U.) ROS (R1) 16.15±4.45 c 18.28±7.40 c 32.04±5.71 c 16.72±4.31 c (R2) 50.08±2.89 b 50.28±8.04 b ±14.98 b ±11.10 b (R3) ±49.77 a ±15.55 a ±16.08 a ±18.36 a 2.5 非特异性酯酶活性和 ROS 含量 5,,, ; 1.2, 5.3 ROS,, ; 2.7, 17.1 (P<0.05) 3 讨论 3.1 鲍类血细胞的分类,, : ( ) [5] [15] (H. discus hannai) Sahaphong [16] (H. asinina) [3] (H. diversicolor supertexa) ; : ;, [2], [4], Travers [17] (H. tuberculata),,, 10% Donaghy [14] (H. discus discus), 3.82% 6.75%,, :, [3],,,,,,, 3.2 鲍类血细胞的组成比例, [5] 55.1% 44.9%; [15] 40% 60%; [3] ( ) ( ) ( ) 3.6% 91.7% 4.7%; 8.55% 58.17% 32.71%,, 55% 6 h 67%,, 36 h 45%; 45% 6 h 33%,, 36 h 55% [18], 3.3 血细胞的结构 [2],,, 12 / 2015 / 39 / 12

6 [3],,,,,, [2-3],, ;, 3.4 血细胞的免疫功能,, [7],,, [12, 19],,, [20],,,, [9, 13-14, 21],, 54.32%~75.17%, 15.74%~28.80%, [9, 14, 21], 20.03%~31.64%,, [22-23],,,,,, FSC-SSC,,,, 86.3% [3],,, (reactive oxygen species, ROS) (O 2 ) ( H 2 O 2 ) ( OH), ROS,, ROS,, [23] ROS [15, 18, 24] ROS, ROS,, ROS : [1],. [J]., 2008, 27(1): [2],,. [J]., 2007, 31( ): [3],,,. [J]. ( ), 2007, 23(3): [4],,,. [J]., 2001, 25(6): [5],,,. [J]., 2008, 27(2): [6],,,. [J]., 2012, 36(2): [7],,. [J]., 2012, 42(1): [8],. [J]., 2013, 37(2): [9] Bado-Nilles A, Gagnaire B, Thomas-Guyon H, et al. Effects of 16 pure hydrocarbons and two oils on haemocyte and haemolymphatic parameters in the Pacific oyster, Crassostrea gigas (Thunberg) [J]. Toxicology in Vitro, 2008, 22(6): [10] Brousseau P, Pellerin J, Morin Y, et al. Flow cytometry as a tool to monitor the disturbance of phagocytosis in the clam Mya arenaria hemocytes following in vitro exposure Marine Sciences / Vol. 39, No. 12 /

7 to heavy metals[j]. Toxicology, 2000, 142: [11] Gagnaire B, Thomas-Guyon H, Renault T. In vitro effects of cadmium and mercury on Pacific oyster, Crassostrea gigas (Thunberg), haemocytes[j]. Fish & Shellfish Immunology, 2004, 16: [12],,. [J]., 2010, 34(9): [13],,,. [J]., 2010, 29(4): [14] Donaghy L, Hong H, Lambert C, et al. First characterisation of the populations and immune-related activities of hemocytes from two edible gastropod species, the disk abalone, Haliotis discus discus and the spiny top shell, Turbo cornutus[j]. Fish & Shellfish Immunology, 2010, 28: [15],,. [J]., 2004, 19(3): [16] Sahaphong S, Linthong V, Wanichano C, et al. Morphofunctional study of the hemocyte of Haliotis asinine[j]. Joumal of Shellfish Research, 2001, 20(1): [17] Travers M, da Silva P M, Goıc N L, et al. Morphologic, cytometric and functional characterisation of abalone (Haliotis tuberculata) haemocytes[j]. Fish & Shellfish Immunology, 2008, 24: [18],,,. [J]., 2010, 29(3): [19],,. (a) [J]., 2011, 30(2): [20] Xian J A, Wang A L, Chen X D, et al. Cytotoxicity of nitrite on haemocytes of the tiger shrimp, Penaeus monodon, using flow cytometric analysis[j]. Aquaculture, 2011, 317: [21] Donaghy L, Kim B, Hong H, et al. Flow cytometry studies on the populations and immune parameters of the hemocytes of the Suminoe oyster, Crassostrea ariakensis[j]. Fish & Shellfish Immunology, 2009, 27: [22],. [J]., 2003, 27(2): [23],. [J]., 2006, 30(5): [24],,. [J]., 1999, 6(3): Classification, structure, and immune functions of abalone (Haliotis diversicolor) hemocytes using a flow cytometric analysis XIAN Jian-an 1, 2, QIAN Kun 2, GUO Hui 3, WANG Dong-mei 1, ZHANG Xiu-xia 1, MIAO Yu-tao 2, PAN Xun-bin 2, WANG An-li 2 (1. Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Haikou , China; 2. Key Laboratory of Ecology and Environmental Science of Guangdong Higher Education Institutes, Guangdong Provincial Key Laboratory for Healthy and Safe Aquaculture, College of Life Science, South China Normal University, Guangzhou , China; 3. Key Laboratory of Aquaculture in South China Sea for Aquatic Economic Animal of Guang-dong Higher Education Institutes, College of Fisheries, Guangdong Ocean University, Zhanjiang , China) Received: Jan., 20, 2015 Key words: Haliotis diversicolor; flow cytometry; hemocyte; immunity Abstract: The classification, structure, and immune functions of abalone (Haliotis diversicolor) hemocytes were analyzed using flow cytometry. Based on the differences of forward light scatter (FSC) and side light scatter (SSC), hemocytes could be divided into three subpopulations, hyalinocyte, small granulocyte, and large granulocyte in the proportions of 32.71%, 58.17% and 8.55%, respectively. The average apoptotic ratio in all hemocytes was 3.76%. The total phagocytic ratio of hemocytes was 63.67%, and the percentages of cells that internalized one, two, and three or more fluorescent beads were 22.31%, 16.39%, and 24.96%, respectively. Mitochondria mass, lysosome mass, non-specific esterase activity, and non-induced reactive oxygen species (ROS) production were highest in the large granulocyte and lowest in the hyalinocyte. These results indicate that structure and immune functions are different between the three hemocyte subpopulations, and that small granulocytes and large granulocytes may play a more important role in the immune response of H. diversicolor. 14 / 2015 / 39 / 12 ( 本文编辑 : 康亦兼 )