拟穴青蟹血细胞原代培养及条件优化

中国畜牧兽医 ›› 2014, Vol. 41 ›› Issue (9): 145-149.

拟穴青蟹血细胞原代培养及条件优化

乔琨¹, 张亚群¹, 王书平¹, 安哲¹, 郝华^1,2, 陈芳奕^1,2, 彭会^1,2

1. 厦门大学海洋与地球学院, 福建厦门 361102;
2. 福建省海洋生物制备技术工程实验室, 福建厦门 361102

修回日期:2014-07-02 出版日期:2014-09-20 发布日期:2014-09-24
通讯作者: 彭会 E-mail:penghui@xmu.edu.cn
作者简介:乔琨（1983- ），女，河北人，博士生，研究方向：海洋分子生物学。
基金资助:
福建省农业科技重点项目（2012N0029）；中国博士后科学基金面上资助（2014M550354）。

The Optimization of Primary Hemocyte Culture of Scylla paramamosain

QIAO Kun¹, ZHANG Ya-qun¹, WANG Shu-ping¹, AN Zhe¹, HAO Hua^1,2, CHEN Fang-yi^1,2, PENG Hui^1,2

1. College of Ocean and Earth Science, Xiamen University, Xiamen 361102, China;
2. Fujian Engineering Laboratory of Marine Bioproducts and Technology, Xiamen 361102, China

Revised:2014-07-02 Online:2014-09-20 Published:2014-09-24

摘要/Abstract

摘要： 本研究通过对拟穴青蟹血细胞原代培养条件的摸索与优化，成功在体外培养了青蟹血细胞。结果表明，pH 4.6的海洋甲壳类血细胞抗凝剂培养青蟹血细胞的形态优于海洋蟹类抗凝剂（ACD）；高血清浓度（>15%）培养血细胞形态较差，易出现空泡；添加5% FBS与1.2% NaCl的L15培养基是培养拟穴青蟹血细胞最适培养基，并可在培养的青蟹原代血细胞中检测到稳定的管家基因表达，表明该细胞可用于拟穴青蟹体外功能试验研究。

关键词: 拟穴青蟹; 血细胞; 原代培养

Abstract: The optimization of primary hemocyte culture of Scylla paramamosain was successfully studied in vitro.The results showed that the hemocyte cells were morphologically kept activity better at pH 4.6 marine crustaceans anticoagulant.High concentration of fetal bovine serum (>15%) in the culture lead to development of vacuoles.The hemocyte cells survived best in L15 medium supplemented with 5% fetal bovine serum and 1.2% sodium chloride.The house keeping gene β-actin of Scylla paramamosain of cultured hemocyte maintained stably expressed through the experiment.This cell culture system could be used for in vitro experiments.

Key words: Scylla paramamosain; hemocyte; primary culture

中图分类号:

Q813.1

乔琨, 张亚群, 王书平, 安哲, 郝华, 陈芳奕, 彭会. 拟穴青蟹血细胞原代培养及条件优化[J]. 中国畜牧兽医, 2014, 41(9): 145-149.

QIAO Kun, ZHANG Ya-qun, WANG Shu-ping, AN Zhe, HAO Hua, CHEN Fang-yi, PENG Hui. The Optimization of Primary Hemocyte Culture of Scylla paramamosain[J]. , 2014, 41(9): 145-149.

参考文献

1 吴宏梅,刘帅,包阿东,等.不同胎牛血清对动物细胞体外培养的影响[J].中国畜牧兽医,2009,36(4):96～99.

2 林琪,李少菁,黎中宝,等.中国东南沿海青蟹属(Scylla)的种类组成[J].水产学报,2007,31(2):211～219.

3 周凯,房文红,乔振国.锯缘青蟹血细胞的形态及分类[J].中国水产科学,2006,13(2):211～216.

4 周婵,朱勇,徐水.无脊椎动物细胞原代培养方法[J].动物营养学报,2011,23(2):203～209.

5 洪宇航,杨筱珍,张金彪,等.中华绒螯蟹血细胞原代培养条件的优化[J].动物学杂志,2012,47(2):52～58.

6 Chen F Y,Liu H P,Bo J,et al.Identification of genes differentially expressed in hemocytes of Scylla paramamosain in response to lipopolysaccharide[J].Fish & Shellfish Immunology,2010,28(1):167～177.

7 Du J,Ou J,Li W,et al.Primary hemocyte culture of the freshwater prawn Macrobrachium rosenbergii and its susceptibility to the novel pathogen spiroplasma strain MR-1008[J].Aquaculture,2012,330:21～28.

8 Ellender R,Najafabadi A,Middlebrooks B L.Observations on the primary culture of hemocytes of Penaeus[J].Journal of Crustacean Biology,1992,12(2):178～185.

9 George S K,Dhar A K.An improved method of cell culture system from eye stalk,hepatopancreas,muscle,ovary,and hemocytes of Penaeus vannamei[J].In vitro Cellular & Developmental Biology-Animal,2010,46(9):801～810.

10 Hoffmann J A.The immune response of Drosophila[J].Nature,2003,426(6962):33～38.

11 Itami T,Maeda M,Kondo M,et al.Primary culture of lymphoid organ cells and haemocytes of kuruma shrimp,Penaeus japonicus[J].Methods in Cell Science,1999,21(4):237～244.

12 Jiang Y S,Zhan W B,Wang S B,et al.Development of primary shrimp hemocyte cultures of Penaeus chinensis to study white spot syndrome virus (WSSV) infection[J].Aquaculture,2006,253(1):114～119.

13 Jiravanichpaisal P,Lee B L,Sderhll K.Cell-mediated immunity in arthropods: Hematopoiesis,coagulation,melanization and opsonization[J].Immunobiology,2006,211(4):213～236.

14 Johansson M W,Keyser P,Sritunyalucksana K,et al.Crustacean haemocytes and haematopoiesis[J].Aquaculture,2000,191(1):45～52.

15 Mangkalanan S,Sanguanrat P,Utairangsri T,et al.Characterization of the circulating hemocytes in mud crab (Scylla olivacea) revealed phenoloxidase activity[J].Developmental & Comparative Immunology,2014,44(1):116～123.

16 Matozzo V,Marin M G.The role of haemocytes from the crab Carcinus aestuarii (Crustacea,Decapoda) in immune responses:A first survey[J].Fish & Shellfish Immunology,2010,28(4):534～541.

17 Nadala E C,Lu Y,Loh P C.Primary culture of lymphoid,nerve,and ovary cells from Penaeus stylirostris and Penaeus vannamei[J].In vitro Cellular & Developmental Biology-Animal,1993,29(8):620～622.

18 Sashikumar A,Desai P V.Development of primary cell culture from Scylla serrata[J].Cytotechnology,2008,56(3):161～169.

19 Shashikumar A,Desai P V.Development of cell line from the testicular tissues of crab Scylla serrata[J].Cytotechnology,2011,63(5):473～480.

20 Shashikumar A,Desai P V.Susceptibility of testicular cell cultures of crab,Scylla serrata (Forskl) to white spot syndrome virus[J].Cytotechnology,2013,65(2):253～262.

21 Sderhll K,Smith V J.Separation of the haemocyte populations of Carcinusmaenas and other marine decapods,and prophenoloxidase distribution[J].Developmental & Comparative Immunology,1983,7(2):229～239.

22 Walton A,Smith V J.Primary culture of the hyaline haemocytes from marine decapods[J].Fish & Shellfish Immunology,1999,9(3):181～194.

23 Xu Y,Ye H,Ma J,et al.Primary culture and characteristic morphologies of neurons from the cerebral ganglion of the mud crab,Scylla paramamosain[J].In vitro Cellular & Developmental Biology-Animal,2010,46(8):708～717.

24 Zeng H,Ye H,Li S,et al.Hepatopancreas cell cultures from mud crab,Scylla paramamosain[J].In vitro Cellular & Developmental Biology-Animal,2010,46(5):431～437.

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