金钱鱼鳃细胞系的建立及其生长特性的初步研究

doi:10.11978/2019017

热带海洋学报 ›› 2019, Vol. 38 ›› Issue (6): 90-97.doi: 10.11978/2019017CSTR: 32234.14.2019017

金钱鱼鳃细胞系的建立及其生长特性的初步研究

周佳楠¹, 苏冒亮^2,³, 张俊彬^1,²()

^1. 上海海洋大学水产科学国家级实验教学示范中心, 上海 201306
^2. 深圳市海洋生物资源与生态环境科学重点实验室, 深圳大学生命科学与海洋学院, 广东深圳 518060
^3. 光电子器件与系统教育部和广东省重点实验室, 深圳大学光电工程学院, 广东深圳 518060

收稿日期:2019-02-18 修回日期:2019-04-03 出版日期:2019-11-20 发布日期:2019-11-26
通讯作者: 张俊彬
作者简介:周佳楠(1995—), 女, 新疆维吾尔自治区昌吉市人, 硕士, 从事海洋生物学研究。E-mail: jiananzhou0608@163.com
基金资助:
国家自然科学基金项目(41806177);国家自然科学基金项目(41741006)

Establishment and characterization of gill cell line from the spotted scat Scatophagus argus

ZHOU Jianan¹, SU Maoliang^2,³, ZHANG Junbin^1,²()

^1. National Demonstration Center for Experimental Fisheries Science Education, Shanghai Ocean University, Shanghai 201306, China
^2. Shenzhen Key Laboratory of Marine Bioresource & Eco-Environmental Science, College of Life Sciences and Oceanography, Shenzhen University, Shenzhen 518060, China;
^3. Key Laboratory of Optoelectronic Devices and Systems of Ministry of Education and Guangdong Province, College of Optoelectronic Engineering, Shenzhen University, Shenzhen 518060, China

Received:2019-02-18 Revised:2019-04-03 Online:2019-11-20 Published:2019-11-26
Contact: Junbin ZHANG
Supported by:
National Natural Science Fund(41806177);National Natural Science Fund(41741006)

摘要/Abstract

摘要：

鳃是鱼类渗透压调节的主要器官, 鳃细胞的培养技术的建立可以为鱼类渗透压调节机理研究提供重要的实验手段和研究方法。金钱鱼(Scatophagus argus)作为一种广盐性鱼类, 是研究渗透压调节机制的理想动物模型。本研究采用胰蛋白酶消化法进行了金钱鱼鳃细胞的体外培养, 确定该类细胞原代和传代培养的最优条件, 分析了其生长特性。结果表明, 金钱鱼鳃细胞系在28℃, 含有20%胎牛血清(fetal bovine serum, FBS)的L-15培养基中生长最佳, 2~4d即可传代, 传代后可稳定培养, 命名为SG。在传代培养过程中, 对其增殖情况进行分析, 发现SG细胞群体倍增时间为40.8h。正常传代的SG细胞冻存、复苏后, 经台盼蓝染色测得细胞存活率约为87%。鳃细胞直接同水体环境接触, 具有高效的渗透压调节能力。环境盐度变化时, 鳃细胞启动渗透压胁迫应答机制, 维持内环境稳态。本研究对SG细胞进行渗透压刺激后, 检测其增殖情况并观察形态变化。分析表明, SG细胞在分别为150和600mOsmol·kg ^-1的低渗和高渗胁迫后均可增殖, 且低渗胁迫后的增殖速度是高渗胁迫的1.5倍。渗透压胁迫后SG细胞的形态观察结果显示, 低渗培养基胁迫后细胞体积发生膨胀而高渗条件下细胞体积发生皱缩。由此推断SG细胞具有较强渗透压耐受性, 且低渗耐受能力强于高渗。SG细胞系的建立为金钱鱼渗透压应答机制和相关基因功能的研究提供了基础实验材料。

关键词: 金钱鱼, 鳃细胞系, 原代培养, 传代培养

Abstract:

Gill is the main osmoregulatory organ in fish, and the establishment of gill cell can provide an important experimental platform to reveal the osmoregulatory mechanism. As a euryhaline fish, Scatophagus argus is an ideal animal model for such studies. This study aimed to establish the branchial cell line of S. argus. The conditions for primary culture and subculture were optimized, and cell characteristics were analyzed. The optimum growth performance was observed in the branchial cell line of S. argus at 28℃ in L-15 that contained 20% fetal bovine serum (FBS), and could be successfully subcultured in 2-4 days. The population doubling time (PDT) of SG (abbreviation for the branchial cell line established in present study) cell line was 40.8 h. Eighty-seven percent SG cells could be resuscitated after cryopreservation in liquid nitrogen. To adapt to the water environment, branchial cells could maintain homeostasis through the osmotic stress response mechanism. SG cells were grown in culture media at different osmotic pressures, and cell proliferation and morphological changes were observed in this study. SG cells could proliferate during hypotonic and hyperosmotic stress (150 and 600 mOsmol·kg ^-1), and the proliferation rate under hypotonic stress was 1.5 times higher than that under hyperosmotic stress. It was observed that the volume of SG cell expanded in the hypotonic medium and shrank in the hyperosmotic condition. Our results indicated that SG cells have strong osmotic tolerance, with a high adaptation in the hypoosmotic environment. The establishment of SG cell line provides basic experimental materials for the study of osmotic pressure response mechanism.

Key words: Scatophagus argus, gill cell line, primary culture, subculture

周佳楠, 苏冒亮, 张俊彬. 金钱鱼鳃细胞系的建立及其生长特性的初步研究[J]. 热带海洋学报, 2019, 38(6): 90-97.

ZHOU Jianan, SU Maoliang, ZHANG Junbin. Establishment and characterization of gill cell line from the spotted scat Scatophagus argus[J]. Journal of Tropical Oceanography, 2019, 38(6): 90-97.

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引物名称	序列(5′—3′)	产物大小/bp
18S rRNA F	GGTAACGGGGAATCAGGGT	819
18S rRNA R	GTGGTGCCCTTCCGTCAA	819

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金钱鱼鳃细胞系的建立及其生长特性的初步研究

Establishment and characterization of gill cell line from the spotted scat Scatophagus argus

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