鳜脑组织细胞系的建立及其病毒敏感性研究

Establishment of a cell line derived from the brain tissue of madanrin fish Siniperca chuatsi and its susceptibility to infection by fish viruses

中文关键词: 鳜  脑组织  细胞系  生物学特性  病毒敏感性

英文关键词: Mandarin fish Siniperca chuatsi  Brain tissue  Cell line  Biological characteristics  Viral susceptibility

中文摘要:

      鱼类细胞是开展鱼类病毒分离鉴定、功能基因分析以及生物制品制备等研究的重要物质基础。鳜(Siniperca chuatsi)是深受养殖者和消费者欢迎的养殖品种。随着鳜鱼养殖产量的逐年增加，其病害问题尤其是病毒病问题也日趋严重，但是，可用于鳜鱼病毒分离和基因功能分析的鳜细胞系缺乏。本研究采用组织块消化法，对来源鳜脑组织的细胞进行原代培养，建立了鳜脑组织细胞系，命名为MFB。MFB细胞在28℃含10%胎牛血清的L-15中已稳定传代超过70次，第25代鳜脑组织细胞的染色体众数为56。采用免疫荧光细胞化学技术(β-tubulin和Neu-N)鉴定MFB细胞的神经元纯度，结果显示，培养的MFB细胞为神经元类细胞。病毒敏感性实验结果显示，鳜蛙虹彩病毒(MFRaIV)、大口黑鲈蛙虹彩病毒(LMBRaIV)和大鲵虹彩病毒(GSIV)均可在MFB细胞中产生典型细胞病变效应，病毒滴度分别为108.68±0.12、108.36±0.15、1010.15±1.85 TCID50/mL。使用脂质体Lipofectamine®2000将pEGFP-N1转入MFB细胞，转染效率可达20%。本研究建立的鳜脑组织细胞系不仅对多种蛙虹彩病毒敏感，而且转染质粒效率较高，为鳜病毒性病原的分离及基因功能研究奠定了前期基础。

英文摘要:

      Fish cells play an important role in virus isolation, identification, functional gene analysis, and biological product preparation. Mandarin fish Siniperca chuatsi is one of the most popular aquaculture species. With a rapid increase in production, the occurrence of diseases has also increased. Moreover, there are very few cell lines of mandarin fish that can be used for virus isolation and gene function identification. In this study, the body surface of mandarin fish was disinfected with povidone-iodine and 75% alcohol. The brain tissue was removed and washed in PBS buffer containing 2´antibiotic-antimycotic 4~5 times in a biosafety cabinet. The tissue was cut into blocks of approximately 3 mm3 with sterilized ophthalmic scissors in a sterile petri dish. After digestion at 37℃ for 0.5~1 h, the tissue blocks were cleaned with L-15 medium containing 10% fetal bovine serum (FBS). The suspension was centrifuged at 1000 r/min for 5 min, and the tissue blocks were transferred into a cell culture flask. A medium containing 30% FBS, penicillin, streptomycin, bFGF, and IGF was added to the cell culture flask, which was placed in an incubator at 28℃ for primary culture to establish the brain tissue cell line. To determine the growth properties of the new mandarin fish brain (MFB) cells, the cells were seeded onto 24-well plates with L-15 medium containing 10% FBS at an initial density of 5×104 cells per well. Cell growth rates were compared under different conditions, including five different media, four different serum concentrations, and four different temperatures. The optimal culture conditions for MFB cells were L-15 containing 10% FBS at 28℃. Under the optimal culture conditions, the doubling time of MFB cell number was ~46.6 h. Chromosome numbers for the 25th generation MFB ranged from 20 to 60, with a mode of 56 and frequency of 20%. Using the total genomic DNA of MFB cells as a template, specific primers were designed for the 28S rRNA gene. A partial gene fragment of 528 bp was obtained by PCR amplification, consistent with the expected size. The PCR-amplified fragments were sequenced and compared with the GenBank database by BLAST analysis. The sequence was consistent with that published in GenBank for the mandarin fish 28S rRNA gene (EF120974). Confirming that the cell line was derived from mandarin fish. NeuN (neuronal nucleus) is a neuro-specific nuclear regulatory molecule and a unique neuronal binding protein that is widely used in the study and diagnosis of neuronal antigens after mitosis. β-tubulin Ⅲ is a signature skeletal protein of mature neurons and is mainly distributed in the synapses and cytoplasm. The purity of MFB cells was determined by immunofluorescence cytochemistry (using β-tubulin and Neu-N). The results showed that all cultured MFB cells were neuron-like. Virus research requires the establishment of a sensitive cell line that allows for the proliferation of viruses in living cells. In this study, MFB cells were used to culture common aquatic viruses. The results of the virus sensitivity test showed that MFRaIV, LMBRaIV, and GSIV could infect and produce typical cytopathic effects in MFB cells, with titers of 108.68±0.12, 108.36±0.15, and 1010.15±1.85 TCID50/mL, respectively; thus, MFB cells are sensitive to MFRaIV, LMBRaIV, and GSIV. The replication level of GSIV in MFB cells was higher than that in MFRaIV and LMBRaIV. MFB cells were insensitive to ISKNV, GCrV-I, KHV, CEV, and CyHV-2. Transfection of exogenous genes into cells is a crucial step in gene function research. After 50 passages, Lipofectamine®2000 was used to transfer pEGFP-N1 into MFB cells. Green cells were observed under a fluorescence inverted microscope 48 h after transfection. The transfection efficiency was determined as the proportion of green positive cells in five random fields; the proportion of green cells was (22.20±1.72)%. In conclusion, a cell line derived from the brain tissue of mandarin fish was successfully established in this study. It is sensitive to a variety of aquatic animal viruses and can be used for gene transfection. It not only enriches the available resources of mandarin fish cells but also provides important experimental materials for further research into infection mechanisms and the development of virus and disease prevention technologies.