牙鲆卵原细胞鉴定、分离及纯化的初步研究  

Identification, Separation and Enrichment of Oogonia from Japanese Flounder (Paralichthys olivaceus)

作　　者：孙朝徽[1] 于清海[1] 周勤[1] 宋立民[1] 姜秀凤[1] 王青林[1] 任建功[1] 王玉芬[1] 任玉芹[1] Sun Zhaohui;Yu Qinhai;Zhou Qin;Song Limin;Jiang Xiufeng;Wang Qinglin;Ren Jiangong;Wang Yufen;Ren Yuqin(Beidaihe Central Experiment Station,Chinese Academy of Fishery Sciences,Qinhuangdao,People＇s Republic of China,Qinghuangdao 066100,China)

机构地区：[1]中国水产科学研究院北戴河中心实验站,秦皇岛066100

出　　处：《中国细胞生物学学报》2018年第9期1510-1517,共8页Chinese Journal of Cell Biology

基　　金：中国水产科学研究院基本科研业务费(批准号:2016PT06);国家水产种质资源共享服务平台运行服务项目(批准号:2018DKA30470);现代农业产业技术体系专项经费(批准号:CARS-47)资助的课题~~

摘　　要：为获得高比例和高数量的卵原细胞,采用酶组合消化和Percoll梯度离心法开展了不同月龄牙鲆卵原细胞分离纯化的研究。通过光学观察及Vasa蛋白在生殖细胞的定位对卵原细胞进行了鉴定区分。结果表明,所获卵巢单细胞悬液中,6月龄每毫克卵巢组织获得的卵原细胞量为(9.86±1.02)×10~4个,显著高于18月龄和30月龄(P<0.05)。Percoll梯度离心后,卵原细胞主要分布于L-15与20% Percoll梯度液间、20%~35% Percoll梯度液间,其占比高达75%以上,显著高于纯化前卵原细胞占比(P<0.05);且细胞存活率高达84%以上,与纯化前细胞存活率无显著性差异(P>0.05)。除18月龄外,6月龄和30月龄每毫克卵巢组织获得的卵原细胞量纯化前后均无显著性差异(P>0.05)。该文通过酶组合消化和Percoll梯度离心的方法对最适月龄—6月龄牙鲆卵原细胞进行分离纯化,最终可获得高比例、高数量和高活性的卵原细胞,为进一步开展牙鲆生殖细胞移植、基因修饰等研究提供了必备的细胞来源。In order to obtain high proportion and plenty of oogonia from Japanese flounder, the differences of isolation of oogonia from the fish of different months of age were compared and analysed by enzyme combination digestion and Percoll gradient centrifugation in this study. In addition, the oogonia were identified by optical observation and immunolocalization of Vasa protein in the germ cells. The results showed that（9.86±1.02）×104 oogonia were obtained from 1 mg of 6-month-old ovaries, which were significantly higher than those of 18 months and 30 months（P〈0.05）. After Percoll gradient centrifugation, the oogonia were mainly distributed in L-15-20% Percoll gradient fluid and 20%-35% Percoll gradient fluid. The proportions of the oogonia of the two cell zones were more than 75%, which were significantly higher than those of the beginning（P〈0.05）. Moreover, the cell survival rates were more than 84%, roughly matched those of the pre-purified cells（P〈0.05）. There were no significantly differences between the number of oogonia before and after purification from 1 mg of 6-or 30-month-old ovaries except for 18-month-old（P〈0.05）. High proportion and abundant oogonia can be obtained from 6-month-old ovary through enzyme combination digestion and Percoll gradient centrifugation, so as to provide the necessary source of cells for further develop germ cell transplantation, gene modification research.

关 键 词：牙鲆 卵原细胞 鉴定 分离 纯化 VASA 

分 类 号：Q952[生物学—动物学]