光唇鱼(Acrossocheilus fasciatus)卵原干细胞提取、冻存及移植研究  

STUDY ON EXTRACTION,CRYOPRESERVATION AND TRANSPLANTATION OF OVOGONIAL STEM CELLS FROM ACROSSOCHEILUS FASCIATUS

作　　者：王宏艳 李文雅 苗亮[1,2] 潘帆 包宁 姜建湖[3] 陈炯[1,2] 李明云[1,2] WANG Hong-Yan;LI Wen-Ya;MIAO Liang;PAN Fan;BAO Ning;JIANG Jian-Hu;CHEN Jiong;LI Ming-Yun(State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agroproducts,Ningbo University,Ningbo 315211,China;Key Laboratory of Aquacultural Biotechnology(Ningbo University),Ministry of Education,Ningbo 315832,China;Zhejiang Institute of Freshwater Fisheries,Huzhou 313001,China)

机构地区：[1]宁波大学农产品质量安全危害因子与风险防控国家重点实验室,浙江宁波315211 [2]宁波大学水产生物技术教育部重点实验室,浙江宁波315832 [3]浙江省淡水水产研究所,浙江湖州313001

出　　处：《海洋与湖沼》2025年第2期442-449,共8页Oceanologia Et Limnologia Sinica

基　　金：浙江省“领雁”项目,2023C02050号(节能节水模块化水产养殖装备研发与应用)。

摘　　要：为更好地保护和开发光唇鱼(Acrossocheilus fasciatus)资源,进行了光唇鱼卵原干细胞提取、冻存和移植研究。结果显示,光唇鱼卵原干细胞呈椭圆形,长径和短径分别为(16.52±0.87)μm、(10.66±0.87)μm。低温冻存光唇鱼卵巢时以10%的DMSO作为保护剂效果最好,从冻存卵巢中提取卵原干细胞的效率可达新鲜卵巢的42.22%±1.02%。以孵化出膜3日龄斑马鱼仔鱼为受体,将提取的光唇鱼卵原干细胞用PKH26荧光染料标记后移植入受体鱼腹腔。荧光显微镜观察显示,移植后1~5 d期间在受体鱼腹腔中有明显荧光信号、并逐渐向腹腔后端背侧迁移。为追踪移植后光唇鱼卵原干细胞数量变化,根据光唇鱼和斑马鱼线粒体ND5基因差异序列建立了检测光唇鱼线粒体ND5拷贝数的绝对荧光定量PCR法。对移植后1~7 d受体鱼的检测显示,移植后1 d时受体鱼中的光唇鱼ND5序列拷贝数最多[(2.51±1.02)×10^(5)copies/100 ng DNA],移植后3 d时显著减少、仅为移植后1 d时的31.08%,5~7 d时ND5数量未出现显著变化。显微镜观察和PCR检测均表明植入的光唇鱼细胞可在受体中存活。研究结果为进一步开展光唇鱼的种质资源保护、生殖细胞发育等研究奠定了基础,也可为其他鲤科鱼类生殖干细胞研究提供参考资料。In order to protect and develop the resources of Acrossocheilus fasciatus,we investigated the extraction,cryopreservation,and transplantation of A.fasciatus oogenous stem cells(AfOSCs).Results showed that AfOSCs were oval,the major and minor axis were(16.52±0.87)µm and(10.66±0.87)µm,respectively.For cryopreservation of A.fasciatus ovary,the 10%dimethyl sulfoxide(DMSO)was the best cryoprotectant,and the AfOSCs extraction efficiency from cryopreserved oval was 42.22%±1.02%compared with the fresh ovarian.The AfOSCs was labeled with PKH26 fluorescent dye and transplanted into the abdominal cavity of 3 days after Danio rerio larvae hatching.At 1~5 day post transplantation(dpt),the PKH26-labeled cells in the recipient larvae were observed in fluorescent microscopy,and the surviving cells migrated toward the posterior dorsal region of abdominal cavity.To detect the quantity changes of transplanted AfOSCs,we developed an absolute quantitative real-time PCR method based on the differences of mitochondrial ND5 sequence between A.fasciatus and D.rerio.At 1 dpt,the A.fasciatus ND5 sequence was(2.51±1.02)×10^(5)copies/100 ng DNA in recipient D.rerio larvaes.The A.fasciatus ND5 copies significantly decreased to(0.78±0.06)×10^(5)copies/100 ng DNA at 3 dpt,and there were no significant difference from 3 dpt to 7 dpt.The results of fluorescent microscopy observation and qRT-PCR detection showed that the transplanted AfOSCs were viable in the recipient D.rerio.Thus,we established oogenous cells extraction,cryopreservation,and transplantation technique for A.fasciatus,which could provide foundation for further studies on genetic resource conservation and germ cell development of A.fasciatus.

关 键 词：光唇鱼(Acrossocheilus fasciatus) 卵原干细胞 冷冻保存 生殖干细胞移植 

分 类 号：Q492[生物学—生理学] S965[农业科学—水产养殖]