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1 日本七鳃鳗外周血细胞显微结构及类淋巴细胞体外培养逄越 1,2 1, 李庆伟 (1., ; 2., ) 摘要 : 以日本七鳃鳗 (Lampetra japonica) 为研究对象, 观察其外周血细胞显微结构和探讨类淋巴细胞原代培养条件采用 Wright 氏和 Giemsa 氏染色法对日本七鳃鳗外周血液有形成分进行显微观察, 可鉴别出红细胞淋巴细胞中性粒细胞单核细胞嗜酸性粒细胞 ; 未发现嗜碱性粒细胞通过 Ficoll 密度梯度离心法和流式细胞仪分选获得高纯度的七鳃鳗类淋巴细胞经条件优化后确定最适培养液 L % FBS(5% 七鳃鳗血清 )+ 0.46% NaCl, 在 18, ph 6.8~7.0 的条件下对日本七鳃鳗类淋巴细胞进行体外培养, 大多数类淋巴细胞半贴壁生长, 细胞状态较好, 最长可以存活近一个月类淋巴细胞的原代培养为日本七鳃鳗细胞体外培养的深入研究提供了实验依据关键词 : 日本七鳃鳗 (Lampetra japonica); 血细胞 ; 类淋巴细胞 ; 细胞培养中图分类号 : Q952, Q253 文献标识码 : A 文章编号 : (2012) (Lampetra japonica),,,, 2009 [1],,, Fujii [2] [3] 1998, 2000 Shintani [4] PU.1/ Spi-B, PU.1/ Spi-B, 2002, Mayer [5] (Petromyzon marinus),,,,, (lymphocyte-like cells) [6] 2007 Ficoll 2009, Cooper [7] VLRA VLRB, T B,,,,,,, 1 材料与方法 1.1 材料 , 7 d, ( 1.092) ; Leibovitz s L15 Sigma ; : ; : : ( , ); (2011J21DW016) : (1975-),,,,,, : , pangyue01@163.com;,, : , liqw@263.net Marine Sciences / Vol. 36, No. 1 /

2 RPMI 1640 M199 Dulbecco s Modified Eagle Media (DMEM) Hyclone, (Fetal Bovine Serum, FBS) Invitrogen Gibco,, (Poly-D-lysine) ; (Wright) (Giemsa) % 10 % 20 %, 100 U/ ml 100 mg/ L , 1 h, 4,, r/min 20 min,, min,, 20 5 g/l mol/l, g/l, 2 μg/cm 2 5 g/l 0.1 g/l, ph 6.8~ 方法 1.2.1,,,, 10 min, Giemsa Wright 45 min,,, % 10 min, 70% 15 min, 3,,, 2 ml, 2 Hank s ml, 3 ml,, 20 min (20, 1200 r/min),, r/min 5 min,, 95% ml 0.4% 0.5 ml,, 4 / () =/ 100%, 200,,, ( =/ 100%) L15 DMEM M199 RPMI 1640, 20%FBS, (6 ) L15 5% 10% 20% 5%, , 0.01%,, , 2 结果 2.1 日本七鳃鳗外周血细胞的显微结构,, 1 :,,, ( 1-1a) (neutrophil): 24 / 2012 / 36 / 1

3 ,, ( 1-1b, 1-2b, 1-3b) (1-4c), 2-3,, :,,,, ( 1-2d, 1-3d) :,,,,,,,,,,, ( 1-3e, 1-5e, 1-6e) :,,,,, ( 1-3f) 1 ( 400) Fig. 1 Microscopic structures of the lamprey peripheral blood cells 1-1~1-6. ; a. ; b.; c.; d.; e.; f. 1-1~1-6. cell types from different fields of microscope; a. erythrocyte; b. segmented neutrophil; c. stab form neutrophil; d. lymphocyte; e. monocyte; f. eosinophil granulocyte 2.2 不同培养液对日本七鳃鳗类淋巴细胞生长的影响 L15 DMEM M199 RPMI1640, 20%FBS,, 1, L15 RPMI1640 M199 DMEM, 20%FBS 0.46%NaCl L15, L 不同浓度的血清对日本七鳃鳗类淋巴细胞生长的影响 L15, 6 1, 2 20% 5%, 5% 10%FBS, Marine Sciences / Vol. 36, No. 1 /

4 , 2.4 不同的生长基质对日本七鳃鳗类淋巴细胞生长的影响 L %FBS %NaCl,, 3,,, 表 1 不同培养液对日本七鳃鳗类淋巴细胞生长的影响 Tab. 1 Effect of medium on primary cell culture of lamprey lymphocyte-like cells (%) (%) 1 L15+20%FBS %FBS M199+20%FBS DMEM+20%FBS L15+20%FBS+0.46%NaCl : () 表 2 不同血清浓度对日本七鳃鳗类淋巴细胞生长的影响 Tab. 2 Effect of serum on primary cell culture of lamprey lymphocyte-like cells (%) (%) 1 5%FBS %FBS %FBS % 表 3 不同生长基质对日本七鳃鳗类淋巴细胞生长的影响 Tab. 3 Effect of substrates on primary cell culture of lamprey lymphocyte-like cells (%) (%) %Poly-Lys 不同温度对日本七鳃鳗类淋巴细胞生长的影响 L %FBS %NaCl,, 7 d, 4 18, 4, 37, 2.6 日本七鳃鳗类淋巴细胞生长形态学观察 L %FBS %NaCl,, 2,,, ( 2-1),, 表 4 不同温度对日本七鳃鳗类淋巴细胞生长的影响 Tab. 4 Effect of temperature on primary cell culture of lamprey lymphocyte-like cells ( ) (%) (%) / 2012 / 36 / 1

5 ,, ( 2-2),,, 1 h,,,,,,,,,,, 3 4 d 1 ( 2-3), 1,,,,,,,, ( 2-4) 2 ( 400) Fig. 2 Primary culture of lamprey leucocyte erythrocyte and lymphocyte-like cells in vitro d ; d ; d ; lamprey leucocyte cultured for 3 days; 2-2. lamprey erythrocyte cultured for 3 days; 2-3. lamprey lymphocyte-like cells cultured for 3 days; 2-4. lamprey lymphocyte-like cells cultured for 1 month 3 讨论,, [8-9], [10-11],,,,,,,,, (Anguilla japonica) [12] (Siniperca chuatsi) [13] (Boleophthalmus pectinirostris) [14] (Carassius auratus cuvieri) [15] (Muraenesax cinereius) [16] (Cichlasoma managuense) [17], [18], [19], [3] Ficoll Percoll-Paque,, Ficoll 1.092,,,, [20],,,,, 3~4,,,, 5~6, Galloway [21] 0.46%, Marine Sciences / Vol. 36, No. 1 /

6 0.46%NaCl, 5% 20%,, ph 7.2~7.4, ph 6.8~7.2 [22], ph,, ph, L %FBS( 5% )+ 0.46%NaCl,, ph 6.8~7.0, 18, 5%CO 2,,, PHA ConA T LPS B,,, T B PHA ConA LPS,, : [1] Nikitina N, Bronner-Fraser M, Sauka-Spengler T. The sea lamprey Petromyzon marinus: a model for evolutionary and developmental biology[j]. Cold Spring Harb Protoc, 2009, 1: 113. [2] Fujii T. Electron microscopy of the leucocytes of the typhlosole in ammocoetes, with special attention to the antibody-producing cells[j]. J Morphol, 1982, 173 (1): [3],,,. [J]., 1998, 18(6): [4] Shintani S, Terzic J, Sato A, et al. Do lampreys have lymphocytes? The Spi evidence[j]. Proc Natl Acad Sci USA, 2000, 97(13): [5] Mayer W E, Uinuk-Ool T, Tichy H, et al. Isolation and characterization of lymphocyte-like cells from a lamprey[j]. PNAS, 2002, 99 (22): [6],,,. [J]., 2008, 43(1): [7] Guo P, Hirano M, Herrin B R, et al. Dual nature of the adaptive immune system in lampreys[j]. Nature, 2009, 459(7248): [8],,,. [J]., 1994, 14(3): [9],,,. [J]., 1992, 12(3): [10],,,. [J]., 1991, 15 (3): [11],,. [J]., 2009, 02: [12],. [J]., 1997, (4): [13],,,. [J]., 1998, 22(1): [14],,. [J]., 2002, 2: [15],. [J]., 1996, 31(1): [16],. [J]., 2003, 38(6): [17],,,. [J]., 2007, 42(6): [18]. [M]. :, 1999, [19],,. [J]., 2001, 36(6): [20] Maule A G, Schreck C B. Changes in numbers of leukocytes in immune or gans of juvenile cobo Salmon after acute stress cortisol treatment[j]. Aqdat Anim Health, 1990, 2(4): [21] Galloway T M. The osmotic pressure and saline content of the blood of Petromyzon fluviatilis[j]. Crichon Research Scholar, 1933, 1: [22],,. [J]., 1996, 2: / 2012 / 36 / 1

7 Microscopic structures of Japanese lamprey peripheral blood cells and culture of lymphocyte-like cells in vitro PANG Yue 1, 2, LI Qing-wei 1 (1. School of Life Sciences, Liaoning Normal University, Dalian , China; 2. College of Life Science and Technology, Dalian University, Dalian , China) Received: Oct., 5, 2010 Key words: Japanese lamprey; haemocyte; lymphocyte-like cells; cell culture Abstract: The microstructure of Japanese lamprey (Lampetra japonica) peripheral blood cells and the cultivation methods of lymphocyte-like cells were explored. The peripheral blood cells of L. japonica were stained by Wright s and Giemsa s staining methods and five major cell types including erythrocyte, lymphocyte, neutrophil granulocyte, monocyte and eosinophil granulocyte, but not basophil were recognized under light microscope. The lymphocyte-like cells were separated successfully with Ficoll-Paque centrifugation and flow cytometry and were cultured in L15 medium with 20% foetal bovine serum (FBS) including 5% L. japonica FBS and 0.46% NaCl. The optimal temperature of cells is 18 and the optimal ph of cells is They can develop attachment as a sheet of cells after 1 hour. Cells have a good condition for nearly a month. The establishment of primary cell cultures of lymphocyte-like cells from L. japonica provides a useful tool for the longevity of the culture and the investigation in cellular biology. ( 本文编辑 : 谭雪静 ) Marine Sciences / Vol. 36, No. 1 /

Microsoft Word 曹伏君_new_.doc

Microsoft Word 曹伏君_new_.doc 45 2 Vol.45, No.2 2014 3 OCEANOLOGIA ET LIMNOLOGIA SINICA Mar., 2014 (Lethrinus erythropterus) * ( 524025) Wright (Lethrinus erythropterus),,,, ; ; ; Q954.56 doi: 10.11693/hyhz20140100011 (Lethrinus erythropterus)