不同倍性泥鳅鳍细胞系的建立及生物学特性分析

ESTABLISHMENT AND CHARACTERIZATION OF FIN CELL LINES FROM DIFFERENT PLOIDY ORIENTAL WEATHERFISH, MISGURNUS ANGUILLICAUDATUS

采用组织块培养法启动二倍体、三倍体和四倍体泥鳅鳍组织细胞原代培养,采用胰蛋白酶消化法传代,目前二倍体、三倍体和四倍体细胞已经分别传至59代、68代和68代。三种细胞均为成纤维细胞样细胞。鳍组织无菌处理方法是:先用质量浓度为10%的碘伏浸泡15min;后用青霉素和链霉素的混合液(500 IU/mL青霉素,500μg/mL链霉素)浸泡30min;原代培养和早期传代培养所用的培养基为DMEM/F12,添加体积分数为20%的胎牛血清、10 ng/mL人碱性成纤维细胞生长因子(bFGF)、20 ng/mLI型胰岛素样生长因子(IGF-I)以及10μg/mL硫酸软骨素。30代以后所用培养基为20%FBS-DMEM/F12。细胞培养在25℃、5%CO2培养箱中。在此条件下,二倍体、三倍体和四倍体的倍增时间分别为48.43h、36.01h和41.45h;二倍体、三倍体和四倍体的特征染色体数目分别为50条、75条和100条;测定的二倍体、三倍体和四倍体细胞及核的体积比分别为1︰1.37︰2.37和1︰1.53︰1.97;细胞经液氮冷冻保存60d后,解冻复苏后测定存活率分别为(80.88±1.38)%、(84.48±1.13)%、(81.57±1.28)%。不同倍性的泥鳅细胞系的建立丰富了鱼类细胞系的种类,为揭示多倍体鱼类生长、遗传等机制打下基础。

Oriental weatherfish （Misgurnus anguillicaudatus） is one of the most popular cultured species in China, Korean peninsula and Japanese archipelago. Primary culture of fin from diploid, triploid and tetraploid oriental weatherfish was performed with tissue explant method. Until now, cells of three tissues have been subcultured to pas-sage 59 for diploid, 68 for triploid, and 68 for tetraploid, respectively. The special and combined aseptic processing method was immersing the fin tissue in 10% povidone-iodine for 15min, and then in penicillin and streptomycin mixture （500 IU/mL penicillin and 500μg/mL streptomycin） for 30min. The medium of primary culture and early subculture was DMEM/F12, supplementing with 20% fetal bovine serum （FBS）, 10 ng/mL basic fibroblast growth factor（bFGF）, 20 ng/mL insulin-like growth factor-I （IGF-I） and 10μg/mL chondroitin sulfate （pH7.2）. The medium after passage 30 was only 20% FBS-DMEM/F12. The condition of cell culture was 25℃ with 5% CO2continuously. The doubling time were 48.43h, 36.01h and 41.45h in diploid, triploid and tetraploid fin cells at the 50th passage, respectively. The feature chromosome number collected from 100 metaphase plates were 2n=50, 3n=75, 4n=100 in diploid, triploid and tetraploid fin cells, respectively. The cell livability of these three kinds of cell were （80.88±1.38）%, （84.48±1.13）% and （81.57±1.28）% when recovered after being stored in liquid nitrogen for 60d at the 40th passage. Establishment of fin cell lines from different ploidy oriental weatherfish in this study enriched the kinds of the fish cell lines, and would lay the foundation for discovering the mechanism of growth and genetics in polyploidy fish.