文章摘要
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 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:广东省教育厅科研处青年创新人才项目(2016KQNCX062,2017KQNCX087);广东海洋大学博士启动基金(R17027,R17021);广东省大学生创新实验项目(CXXL2016012);广东海洋大学“海之帆”起航计划项目(qhjh2017zr12)
作者单位E-mail
康恺 广东海洋大学 农学院 kangkai610@126.com 
吴江 广东海洋大学 农学院  
苑麟勇 广东海洋大学 农学院  
刘丽欢 广东海洋大学 农学院  
陈永灵 广东海洋大学 农学院  
效梅 广东海洋大学 农学院  
安立龙 广东海洋大学 农学院 anlilong@126.com 
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中文摘要:
      分离培养尼罗罗非鱼精巢支持细胞(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(new 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2d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1~2d,细胞贴壁;培养3~5d,细胞完全贴壁并迅速增殖。单个SCs呈不规则多边形,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% NBS、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)。与无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)。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 was establishment and optimization of isolation and culture system for Sertoli cells of Nile tilapia. Fresh testis of Nile tilapia in the Ⅲ development stage were abtained, and rinsed with phosphate-buffered saline. Then the testis were dissected into segments and digested with 0.5mg/mL collegen for 30minfollowed 0.25% tripsin-0.04%EDTA for 5min, the digestion was terminated with L-15 culture medium with 10% new bovine serum. Sertoli cells were selected according to the characteristic that sertoli cells adhered more qiuckly than germ cells. Sertoli cells were cultured in 96-well plates, and respectively cultured in L-15, M199, or F12 culture medium supplemented with 10%NBS, or L-15 culture medium supplemented with 5%, 10%, 15% new bovine serum, or L-15 culture medium supplemented with 10% new bovine serum and 1% Nile tilapia serum. Sertoli cells in six wells of every group were taken every two days, cell number of every well was counted by haemacytometer, and growth curve of sertoli cells was drawed. Compared with F12 or M199 culture medium, growth of sertoli cells in L-15 culture medium was more qiuckly(P<0.01). Compared with supplemented with 5% or 15% new bovine serium, proliferated of sertoli cells was speeded up (P<0.05) by supplementation of 10% new bovine serium in culture medium. Comparatively, the effect of addition of 1% Nile tilapia serum was greater (P<0.05) than that of without Nile tilapia serium. The proliferation of Nile tilapia Sertoli cells of serum could be improved by supplementation of 10% new bovine serium and 1% Nile tilapia serum in L-15 cell culture medium.
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