Xu Xiaohui, Duan Hu, Shi Yingli, Xie Shijun, Song Zhan, Jin Songjun, Li Fuhua, Xiang Jianhai
Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, 7 Nanhai Rd., Qingdao 266071, China; Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science and Technology, Qingdao, China.
College of Marine Life Sciences, Ocean University of China, Qingdao, China.
Dev Comp Immunol. 2018 Nov;88:45-54. doi: 10.1016/j.dci.2018.07.006. Epub 2018 Jul 9.
Various known and unknown viral diseases can threaten crustacean aquaculture. To develop prophylactic and therapeutic strategies against viruses, crustacean cell lines are urgently needed for immunology and virology studies. However, there are currently no permanent crustacean cell lines available. In this study, we developed a new method for preparing crayfish plasma (CP) and found that CP enhanced the proliferative capacity of haematopoietic tissue (hpt) cells from Cherax quadricarinatus by an EdU (5-ethynyl-2'-deoxyuridine) assay. The optimal CP concentration for hpt cell culture and the optimal subculture method are discussed. To achieve efficient expression of a foreign gene in hpt cells cultured in vitro, different transfection methods and vectors were analysed. We found that Lipofectamine 2000 could be used to efficiently transfect a foreign vector into hpt cells and exhibited a lower level of cytotoxicity than the other methods tested, and transfection of pEGFP-N1/w249 and pDHsp70-EGFP-FLAG resulted in high EGFP expression. By transmission electron microscopy (TEM) and virus copy number analysis, we found that white spot syndrome virus (WSSV) could infect hpt cells and multiply efficiently. Our results implied that the crayfish hpt cell culture system we improved could be used as a replacement for immortal crustacean cell lines in viral infection studies. Our findings provide a solid foundation for future immortalization and gene function studies in crustacean cells.
各种已知和未知的病毒性疾病都会对甲壳类水产养殖构成威胁。为了制定针对病毒的预防和治疗策略，免疫和病毒学研究迫切需要甲壳类细胞系。然而，目前尚无可用的永久性甲壳类细胞系。在本研究中，我们开发了一种制备小龙虾血浆（CP）的新方法，并通过EdU（5-乙炔基-2'-脱氧尿苷）检测发现CP增强了四脊扁水虱造血组织（hpt）细胞的增殖能力。讨论了hpt细胞培养的最佳CP浓度和最佳传代方法。为了在体外培养的hpt细胞中实现外源基因的高效表达，分析了不同的转染方法和载体。我们发现Lipofectamine 2000可用于将外源载体高效转染到hpt细胞中，并且与其他测试方法相比具有较低的细胞毒性，转染pEGFP-N1/w249和pDHsp70-EGFP-FLAG可导致高水平的EGFP表达。通过透射电子显微镜（TEM）和病毒拷贝数分析，我们发现白斑综合征病毒（WSSV）可以感染hpt细胞并高效增殖。我们的结果表明，我们改进的小龙虾hpt细胞培养系统可在病毒感染研究中替代永生化甲壳类细胞系。我们的发现为未来甲壳类细胞的永生化和基因功能研究奠定了坚实的基础。
