Classification of circulating hemocytes from the red swamp crayfish Procambarus clarkii and their susceptibility to the novel pathogen Spiroplasma eriocheiris in vitro
Classification of circulating hemocytes from the red swamp crayfish Procambarus clarkii and their susceptibility to the novel pathogen Spiroplasma eriocheiris in vitro
复制标题
红沼泽克氏原螯虾循环血细胞的分类及其对新病原体鹅掌螺原体的体外敏感性
DOI：
10.1016/j.aquaculture.2012.04.042
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发表时间：
2012-08-01
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：
Ding, Zhengfeng;Du, Jie;Wang, Wen
采用形态学观察和流式细胞术（FCM）将克氏原螯虾（Procambarusclarkii）的血细胞分为透明细胞（H）、半粒细胞（SG）和粒细胞（G）3个亚群。建立了原代血细胞培养体系，用于研究从自然感染的克氏螺旋体中分离的绒螯螺原体的体外增殖。格雷斯昆虫培养基补充有15%胎牛血清（FBS），沿着有100 U ml（-1）青霉素，100 μ g ml（-1）链霉素，最终pH为7.2-7.4，在28 ℃下孵育，支持原代培养物的最佳存活。然而，粒细胞在培养基中迅速裂开，可能影响其他细胞类型的存活。采用Percoll两步密度梯度离心法分离血细胞。当单独培养时，与粒细胞相比，透明质细胞和半粒细胞在孵育16天后保持较高的活力（>75%），粒细胞在2-6天内降解。在挑战S之后。在绒螯蟹中，早在接种后48 h就观察到培养的血细胞的细胞病变效应（CPE），并且随着感染的进行，CPE变得更加明显，在接种的培养物中具有细胞碎片和细胞渗出物。在攻击后60 h内细胞溶解明显。采用实时荧光定量RT-PCR方法检测攻毒过程中2 h、4 h、8 h、10 h、24 h、48 h和60 h的免疫应答情况。从克氏原螯虾血细胞中分离到的过氧化物酶蛋白基因（Peroxinectin，简称Pcpxin）和热休克蛋白70（Heat shock protein 70，HSP 70）基因在S.绒螯蟹挑战以上结果对促进S.研究内容包括阐明发病机理、寄主与病原菌的相互作用及防御机制，并最终导致这一甲壳动物疾病的预防。(C)2012年爱思唯尔B。V.保留所有权利。
Hemocytes from the red swap crayfish Procambarus clarkii were classified into three subpopulations: hyalinocytes (H), semigranulocytes (SG) and granulocytes (G) by morphologic observation and flow cytometry (FCM). The primary hemocyte culture was then established for studies on the in vitro propagation of Spiroplasma eriocheiris isolated from naturally infected P. clarkii. Grace's insect medium supplemented with 15% fetal bovine serum (FBS), along with 100 U ml(-1) penicillin, 100 mu gml(-1) streptomycin, with a final pH of 7.2-7.4, incubated at 28 degrees C, supported the best survival in primary cultures. However, the granulocytes dehisced rapidly in culture medium, potentially impacting the survival of the other cell types. A two-step density gradient centrifugation with Percoll was developed to separate the hemocytes. When cultured separately, hyalinocytes and semigranulocytes maintained higher viability (>75%) after 16 days of incubation compared with granulocytes, which degraded over 2-6 days. After a challenge with S. eriocheiris, cytopathic effects (CPE) of the cultured hemocytes were observed as early as 48 h post-inoculation, and as the infection progressed, CPE became more apparent, with cell debris and cellular exudates in inoculated cultures. Cell lysis was noticeable within 60 h after challenging. A quantitative real-time RT-PCR was conducted to detect the immune responses during the challenging process at 2 h, 4 h, 8 h, 10 h, 24 h, 48 h and 60 h, respectively. Clear time-dependent expression patterns of the peroxinectin gene (referred as Pcpxin), recently isolated from the crayfish hemocytes, and heat shock protein 70 (HSP70) gene were observed after S. eriocheiris challenge. The results above should be helpful in promoting research with S. eriocheiris, including elucidation of pathogenesis, host pathogen interaction and the defense mechanisms, and ultimately lead to prevention of this crustacean disease. (C) 2012 Elsevier B. V. All rights reserved.