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| 罗氏沼虾神经细胞的原代培养及其在病毒研究中的应用 |
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黄笑,王国浩,董宣,唐琼英,段虎,杨国梁,黄倢
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1.湖州师范学院 浙江省水生生物资源养护与开发技术研究重点实验室 中国水产科学研究院水生动物繁育
与营养重点实验室 浙江 湖州 313000;2.中国水产科学研究院黄海水产研究所 青岛海洋科技中心海洋渔业科学
与食物产出过程功能实验室 农业农村部海水养殖病害防治重点实验室 青岛市海水养殖流行病学与
生物安保重点实验室 山东 青岛 266071;3.江苏数丰水产种业有限公司 江苏 高邮 225654;4.天津科技大学海洋与环境学院 天津 30045
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| 摘要: |
| 细胞平台是研究病原–宿主互作的重要工具，虾类细胞系的缺少严重制约了虾类病毒学研究。传染性早熟病毒(IPV)是近年来发现的能够导致罗氏沼虾(Macrobrachium rosenbergii)性早熟和生长缓慢的一种新病毒，主要侵染罗氏沼虾神经组织。为建立罗氏沼虾病毒–宿主互作的体外研究平台，本研究采用木瓜蛋白酶消化健康罗氏沼虾的脑、眼柄、胸神经节和腹神经节，获取罗氏沼虾神经细胞，并利用L-15培养基进行离体培养。选择培养效果最佳的脑神经细胞进行IPV体外感染。结果显示，培养的原代细胞在含有谷氨酰胺的L-15培养基中生长良好，其中，从脑组织和眼柄X器官–窦腺复合体中分离出的细胞在体外存活时间可达15 d，从胸腹神经节中分离出来的细胞在体外存活时间达9 d。在本研究所采用的感染方式和剂量条件下，脑细胞在感染96 h后检测到高载量的IPV。本研究建立了一种操作简便的罗氏沼虾神经细胞原代培养技术，为罗氏沼虾神经内分泌研究和病毒–宿主互作提供了基础数据和平台。 |
| 关键词: 罗氏沼虾 神经细胞 原代培养 传染性早熟病毒 |
| DOI：10.19663/j.issn2095-9869.20230316002 |
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| Primary culture of Macrobrachium rosenbergii neural cells and its application in virus research |
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HUANG Xiao1,2,3,4,5,6,7, WANG Guohao4,5,6,7, DONG Xuan1,2,3,4,5,6,7, TANG Qiongying1,2,3,8, DUAN Hu9, YANG Guoliang1,2,3,8, HUANG Jie4,5,6,7
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1.Zhejiang Provincial Key Laboratory of Aquatic Resources Conservation and Development;2.Key Laboratory of Aquatic Animal Genetic Breeding and Nutrition, Chinese Academy of Fishery Sciences;3.Huzhou University, Huzhou 313000, China;4.Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences;5.Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao Marine Science and Technology Center;6.Key Laboratory of Maricultural Organism Disease Control, Ministry of Agriculture and Rural Affairs;7.Qingdao Key Laboratory of Mariculture Epidemiology and Biosecurity, Qingdao 266071, China;8.Jiangsu Shufeng Prawn Breeding Co. Ltd., Gaoyou 225654, China;9.College of Marine and Environmental Science, Tianjin University of Science and Technology, Tianjin 300457, China
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| Abstract: |
| Macrobrachium rosenbergii is one of the most popular species in aquaculture. However, the M. rosenbergii farming industry has been facing an ongoing iron prawn syndrome (IPS) crisis since 2010, resulting in substantial economic losses to the farming industry. Infectious precocity virus (IPV) is a novel virus of Flaviviridae found in recent years that can cause sexual precocity and associated slow growth in healthy M. rosenbergii. It is believed to have a specific correlation with IPS. There are very few cell lines of crustaceans that can be used for studying the response of cells to pathogens. Even though the primary culture technology of blood and muscle cells in M. rosenbergii has gradually matured, there have been few studies on the primary culture of neural cells. Quantitative results of different tissues of IPV-positive M. rosenbergii have indicated that nerve-rich tissues, such as eyestalk, brain, and thoracic ganglion tissues, have a higher viral load, which explains why IPV has neurotropic tissue characteristics. To provide an in vitro cell platform for studying the virus-host interactions of IPV, a simple, stable, and feasible primary culture method was established for the nervous tissue primary cells of M. rosenbergii.
In this study, healthy prawns with a body length of about 10–12 cm, healthy appendages, and vitality were selected for the experiments. The body surface of M. rosenbergii was first disinfected with 75% alcohol. On the clean bench, the nervous tissues, including the brain, X organ-sinus gland complex from the eyestalk, thoracic ganglion, and abdominal ganglion tissues, were isolated and washed in PBS buffer containing 100 U/mL penicillin and streptomycin, 100 U/mL amphotericin, and 80 U/mL gentamicin, 2–3 times. The tissues were then placed in 5 mL of 0.5% papain solution. After digestion at 25 ℃ for 5 min, 1× L-15 medium containing 15% FBS, 140 mmol/L D-glucose, and antibiotics (100 U/mL penicillin and streptomycin, 100 U/mL amphotericin, and 80 U/mL gentamicin) were added to terminate digestion. Next, the cells were seeded onto a 24-well plate and allowed to settle in darkness at 25 ℃ for 45 min. After adhesion, cells were transferred to a cell incubator and cultured in the dark at 28 ℃. The nervous tissues’ primary cells at different time points were observed under an inverted microscope, and the morphological changes were recorded through imaging. The compound eye tissue of IPV-positive M. rosenbergii was placed in SM buffer, and the IPV mixture was obtained after multiple rounds of crushing and centrifugation. The brain tissue primary cells with large quantities and better culture effects were selected for the IPV challenge after cultured in vitro for 5 days. The virus crude extract was filtered through a 0.22 μm filter membrane and mixed according to the volume ratio of virus crude extract to serum-free medium = 1∶9. For the experimental group, 2 mL of the mixed solution was added to each well, while the control group received 2 mL of serum-free medium per well. Samples were taken at 0, 6, 12, 24, 48, and 96 h after virus infection. RT-qPCR was used to detect the IPV load.
The results showed that the cultured primary cells grew well in a 1× L-15 medium containing glutamine and serum. According to the morphology of the cells, number of axons, and other characteristics, brain tissue primary cells, primary cells from the eyestalk X organ-sinus gland complex, and thoracic ganglion tissue primary cells were divided into four types: neurosecretory cells, pseudounipolar neural cell-like, bipolar neural cell-like, and multipolar neural cell-like, respectively. In comparison, only round neural cells and bipolar-like neural cells were found in the primary cell culture of the abdominal ganglion tissue under in vitro culture. The brain tissue primary cells and X organ-sinus gland complex cells survived for 15 days, while the thoracic ganglion and abdominal ganglion tissue primary cells survived for 9 days after being cultured in vitro. After IPV infected the brain tissue primary cells, the viral load of IPV was 15.30 copies/μg RNA at 6 h post-infection (hpi) and 35.59 copies/μg RNA at 12 hpi. IPV was not detected in IPV-infected brain tissue primary cells at 24 hpi and 48 hpi. Then, IPV viral load was detected at 96 hpi, reaching 104 copies/μg RNA.
In conclusion, this study successfully established a simple and convenient primary culture technology for cells from M. rosenbergii neural tissues, providing preliminary data and a platform for neuroendocrine and virus-host interactions research. An in vitro infection model of IPV was also initially established in this study. Before establishing the shrimp cell line, the cells could be used to study the mechanism of viral infection, replication, and transcription, providing primary data and a platform for studying the norovirus and neuroendocrine factors of M. rosenbergii. They could also provide critical experimental materials for further research into infection mechanisms and the development of virus and disease prevention technologies. |
| Key words: Macrobrachium rosenbergii Neural cells Primary culture Infectious precocity virus |
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