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

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。

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 stageⅢwere 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.