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

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

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.