Primary hemocyte culture of the freshwater prawn Macrobrachium rosenbergii and its susceptibility to the novel pathogen spiroplasma strain MR-1008
Primary hemocyte culture of the freshwater prawn Macrobrachium rosenbergii and its susceptibility to the novel pathogen spiroplasma strain MR-1008
复制标题
罗氏沼虾原代血细胞培养及其对新型病原菌螺原体MR-1008的敏感性
DOI：
10.1016/j.aquaculture.2011.12.003
复制
发表时间：
2012-02-17
期刊：
影响因子：
4.5
通讯作者：
Wang, Wen
中科院分区：
文献类型：
--
作者：
Du, Jie;Ou, Jiangtao;Wang, Wen
利用罗氏沼虾（Macrobrachium rosenbergii）的血细胞原代培养，研究了新型天然病原菌螺旋体MR-1008的体外繁殖。在这种情况下，对这种甲壳类动物的血细胞培养方法进行了改进，采用Leibovitz-15（L-15）生长培养基，培养基中添加15%胎牛血清（FBS）、0.1%葡萄糖、0.5% NaCl和抗生素（100 μ ml（-1）青霉素、100 μ ml（-1）链霉素、1 μ g ml两性霉素b）， pH值为7.20-7.40。在这种培养基中，血细胞可以传代培养两次，包括显著的细胞增殖和生存能力长达12天。用螺旋体MR-1008攻毒研究血细胞的敏感性。早在接种后48 h就观察到细胞病变效应（CPE）；随着感染的进展，CPE变得更加明显，接种培养物中存在细胞碎片和细胞渗出物。感染后60h细胞出现明显的裂解。分别在感染2、4、12、24、48和60 h时，采用实时定量RT-PCR检测免疫相关基因的表达情况。脂多糖、β -1,3-葡聚糖结合蛋白（LGBP）、酚氧化酶原（proPO）、α -2-巨球蛋白（α 2M）和铜/锌超氧化物歧化酶（Cu/Zn-SOD）基因在罗氏m.s robergii血细胞中的表达水平表明，这些免疫基因是由MR-1008螺旋体攻击诱导的。原代血细胞培养可用于体外研究宿主与病原体之间的相互作用，促进对螺旋体MR-1008的了解，并最终预防这种甲壳类疾病。(C) 2011 Elsevier B.V.版权所有
Primary culture of hemocyte from the giant freshwater prawn, Macrobrachium rosenbergii, was established for studies on the in vitro propagation of the novel nature pathogen spiroplasma MR-1008. In this context, a modified method of hemocyte culture from this crustacean has been standardized by employing Leibovitz-15(L-15) growth medium supplemented with 15% Fetal Bovine Serum (FBS) along with 0.1% glucose, 0.5% NaCl and antibiotics (100 U ml(-1) penicillin, 100 U ml(-1) streptomycin,1 mu g ml amphotericin b) and a suitable pH of 7.20-7.40. In this medium, the hemocytes can be subcultured twice, including significant cell proliferation and viability of up to 12 days. Susceptibility of the hemocytes was investigated by challenging with spiroplasma MR-1008. Cytopathic effects (CPE) were observed as early as 48 h post-inoculation; and as the infection progressed, CPE became more apparent, with cell debris and cellular exudates present in inoculated cultures. Cell lysis was noticeable within 60 h after infection. A quantitative real-time RT-PCR was conducted to detect the immune related gene expression during the infection process at 2, 4, 12, 24, 48, and 60 h, respectively. The expression levels of lipopolysaccharide and beta-1,3-glucan-binding protein (LGBP), prophenoloxidase (proPO), alpha-2-macroglobulin (alpha 2M) and copper/zinc SOD (Cu/Zn-SOD) genes in hemocytes of M. rosenbergii demonstrated that these immune genes were induced by challenge with spiroplasma MR-1008. The primary hemocyte culture can be used to investigate the interaction between host and pathogen in vitro, promote enhanced understanding of spiroplasma MR-1008, and ultimately lead to prevention this crustacean disease. (C) 2011 Elsevier B.V. All rights reserved.