中华鲟精巢细胞系的建立和鉴定  

ESTABLISHMENT AND CHARACTERIZATION OF A TESTICULAR CELL LINE FROM CHINESE STURGEON(ACIPENSER SINENSIS)

作　　者：叶欢[1] 王艺舟 杜浩[1] 岳华梅[1] 阮瑞[1] 罗江[1] 李创举[1] YE Huan;WANG Yi-Zhou;DU Hao;YUE Hua-Mei;RUAN Rui;LUO Jiang;LI Chuang-Ju(Key Laboratory of Freshwater Biodiversity Conservation,Ministry of Agriculture and Rural Affairs,Yangtze River Fisheries Research Institute,Chinese Academy of Fishery Sciences,Wuhan 430223,China;College of Animal Science,Guizhou University,Guiyang 550025,China)

机构地区：[1]中国水产科学研究院长江水产研究所,农业农村部淡水生物多样性保护重点实验室,武汉430223 [2]贵州大学动物科学学院,贵阳550025

出　　处：《水生生物学报》2023年第8期1261-1268,共8页Acta Hydrobiologica Sinica

基　　金：国家重点研发计划(2021YFD1200301和2018YFD0901205);国家自然科学基金(31902353);中国水产科学研究院基本科研业务费专项(2020TD08)资助。

摘　　要：为了开展中华鲟(Acipenser sinensis)种质资源保存及其精原干细胞体外培养的研究,采用蛋白酶消化法,对中华鲟精巢组织细胞进行原代培养,建立了中华鲟精巢细胞系(Acipenser sinensis testicular cell line,AST),经352d传代培养,已稳定传至80代。中华鲟精巢细胞系形态主要呈类纤维状,培养基为DMEM,培养温度为25℃,最适血清浓度为15%。正常传代的AST细胞冻存、复苏后,经台盼蓝染色,约(81.36±1.13)%的细胞具有活性,复苏后细胞仍生长旺盛。染色体核型分析结果显示,第30代中华鲟精巢细胞系染色体数目分布在142—310,众数为264。通过RT-PCR检测发现,在P0和P1细胞中,Sertoli细胞特异表达基因(amh和gsdf)、Leydig细胞特异表达基因(cyp17a1)和生殖细胞特异表达基因(dazl、dnd和vasa)都有表达,且表达量与精巢中的相似;在P15、P30和P60细胞中,只有amh和vasa基因有微弱的表达,说明细胞系传代到了后期,只含有极少量的Sertoli细胞和生殖细胞。通过脂质体转染法将pEGFP-N3质粒转入AST细胞中,可表达增强型绿色荧光蛋白(Enhanced green fluorescent protein,EGFP)。AST细胞系的建立为中华鲟种质资源的保存、精原干细胞的体外增殖与分化、基因功能等研究提供了重要的实验材料。The Chinese sturgeon,Acipenser sinensis,is a large anadromous fish,with an average 14 years for males and 21 years for females.It was listed as critically endangered by the International Union for Conservation of Nature(IUCN)since 2010,and its spawning activity was not observed during the past five spawning seasons.Therefore,there is an urgent need to preserve the genetic resources of Chinese sturgeon.To investigate the germplasm conservation of Chinese sturgeon and its spermatogonia stem cell culture in vitro,a new cell line(designated as AST)derived from the testis of Chinese sturgeon was established and characterized.The AST cell line are fibroblast-like cells,and have been stably subcultured for more than 80 passages.The optimal growth conditions for the AST cell line are DMEM medium,25℃,and 15%FBS.After cryopreservation in liquid nitrogen,the viability of AST cells was about(81.36±1.13)%,as measured by trypan blue staining.Chromosome analysis of AST cells at the 30th passage showed that the number of chromosomes ranged from 142 to 310,and the modal number was 264.To detect the expression characterization of Sertoli cell-related genes(amh and gsdf),Leydig cell-related genes(cyp17a1),and germ cell-related genes(dazl,dnd and vasa)in AST cells by RT-PCR,all of the genes were found in the P0 and P1 cells,where their amounts were similar with those of in testis;however,in P15,P30,and P60 AST cells,only amh and vasa genes were detected with very low level,suggesting only a few number of Sertoli cells and germ cells in the late passage of AST cells.The signal of enhanced green florescence protein(GFP)were detected in a few of AST cells after transfection with pEGFP-N3 plasmid.The establishment of the AST cell line could provide important experimental material for the conservation genetic resources of Chinese sturgeon,the in vitro study of proliferation and differentiation of spermatogonia stem cell,and function analysis of testicular related genes.

关 键 词：精巢 AST细胞系 传代培养 核型分析 基因表达特征 中华鲟 

分 类 号：Q172[生物学—水生生物学]