文章摘要
康恺,吴江,苑麟勇,刘丽欢,陈永灵,效梅,安立龙.尼罗罗非鱼精巢支持细胞分离培养体系建立及优化.渔业科学进展,2020,41(1):119-126
尼罗罗非鱼精巢支持细胞分离培养体系建立及优化
Establishment and optimization of an isolation and culture system for Sertoli cells of Nile tilapia
投稿时间:2018-11-20  修订日期:2018-12-24
DOI:
中文关键词: 尼罗罗非鱼  支持细胞  分离培养  体系优化
英文关键词: Nile tilapia  Sertoli cell  Isolated culture  System optimization
基金项目:
作者单位
康恺 广东海洋大学农学院 湛江 524088 
吴江 广东海洋大学农学院 湛江 524088 
苑麟勇 广东海洋大学农学院 湛江 524088 
刘丽欢 广东海洋大学农学院 湛江 524088 
陈永灵 广东海洋大学农学院 湛江 524088 
效梅 广东海洋大学农学院 湛江 524088 
安立龙 广东海洋大学农学院 湛江 524088 
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中文摘要:
      本研究分离培养尼罗罗非鱼(Oreochromis niloticus)精巢支持细胞(Sertoli cells, SCs),建立并优化尼罗罗非鱼精巢支持细胞分离培养体系。无菌条件下,取发育至第Ⅲ期的尼罗罗非鱼精巢,PBS清洗后,剪碎精巢组织,0.25%胰蛋白酶消化,用含10%犊牛血清(Newborn bovine serum, NBS)的L-15培养液终止消化,过滤、离心,获得细胞。差速贴壁法获得SCs后,在26℃、无CO2饱和湿度恒温培养箱中培养。分别采用含10% NBS的L-15、M199、F12培养液,含5%、10%、15% NBS的L-15培养液,含1%罗非鱼血清的L-15+10% NBS培养液培养尼罗罗非鱼SCs。各组每2 d取细胞计数,绘制SCs生长曲线;甲基绿染色,倒置显微镜下观察SCs的形态。尼罗罗非鱼SCs培养1~2 d,细胞贴壁;培养3~5 d,细胞完全贴壁并迅速增殖。单个SCs呈不规则多边形,其核位于胞质中央,呈卵圆形,胞质中可见吞噬颗粒和空泡,空泡聚集于支持细胞胞质的两极或散布于核的四周。SCs在L-15培养液中贴壁生长,在F12、M199培养基中较难贴壁。与F12、M199培养液相比,L-15培养液更有助于尼罗罗非鱼SCs生长增殖(P<0.01)。与添加5%和15% NBS相比,在L-15培养基中添加10% NBS更有助于尼罗罗非鱼SCs生长增殖(P<0.05)。与未添加尼罗罗非鱼血清相比,在10% NBS+L-15培养中添加1%尼罗罗非鱼血清,能显著促进SCs生长增殖(P<0.05)。研究表明,采用胰酶消化差速贴壁法获得尼罗罗非鱼精巢支持细胞,10% NBS+1%罗非鱼血清+L-15培养液培养,能显著促进尼罗罗非鱼精巢支持细胞生长增殖。
英文摘要:
      The aim of this study to establish and optimize an isolation and culture system for Sertoli cells of Nile tilapia. Fresh testis tissues from Nile tilapia in development stage Ⅲ were obtained and then rinsed with phosphate-buffered saline. The tissue was dissected into segments and digested with 0.5 mg/ml collagen for 30 min, followed by 0.25% trypsin–0.04% EDTA for 5 min, and the digestion was terminated with L-15 culture medium with 10% newborn bovine serum (NBS). Sertoli cells were selected using the characteristic that they adhered more quickly than germ cells to the culture vessels. Sertoli cells were cultured in 96-well plates in L-15, M199, or F12 culture medium, supplemented with 10% NBS, or L-15 culture medium supplemented with 5%, 10%, and 15% NBS, or L-15 culture medium supplemented with 10% NBS and 1% Nile tilapia serum. For each treatment group, Sertoli cells were collected from six culture wells every 2 days; the number of cells in each well was counted using a hemocytometer, and a growth curve was drawn for the Sertoli cells. Compared with F12 or M199 culture medium, more rapid growth of Sertoli cells occurred in the L-15 culture medium (P<0.01). Compared with supplementation with 5% or 15% NBS, the proliferation of Sertoli cells was accelerated (P<0.05) by supplementation with 10% NBS in the culture medium. Comparatively, the effect of the addition of 1% Nile tilapia serum was greater (P<0.05) than the absence of serum. The proliferation of Nile tilapia Sertoli cells can be improved by supplementation with 10% NBS and 1% Nile tilapia serum in L-15 cell culture medium.
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