大黄鱼性腺原代细胞培养技术的建立

Establishment of Primary Cell Culture Technique for Gonadal Tissue of Large Yellow Croaker

为建立大黄鱼精巢和卵巢组织的原代细胞培养技术,获得单层的性腺原代细胞,采用组织块贴壁法,对来源于大黄鱼的卵巢和精巢组织的细胞进行原代培养,并使用0.25%胰酶消化法尝试传代培养。在26℃条件下,添加20%胎牛血清,在100 IU/mL青霉素(Penicillin)和100μg/mL硫酸链霉素(Streptomycin)的DMEM/F-12(HAM)1∶1培养基中培养大黄鱼卵巢和精巢组织的细胞。结果显示:1)大黄鱼卵巢组织在贴壁后第4天成纤维细胞开始迁出,呈辐射状生长;贴壁后第6天迁移出上皮样细胞,呈“铺石路”状生长;贴壁后第17天左右出现不同程度贴壁抑制。2)大黄鱼精巢组织在贴壁后第5天迁出成纤维细胞;第9~17天,部分成纤维细胞呈长梭形涡旋状生长,部分呈三角形均匀分布增殖;贴壁后第18天左右部分细胞空泡化严重。3)在培养后第8天对卵巢和精巢组织原代细胞进行胰酶消化传代培养,传代培养第2天部分细胞贴壁分裂增殖,传代培养3 d后可进行第二次传代,第二次传代后细胞长势变缓,细胞空泡化加快。

In order to establish the primary cell culture technique of the testis and ovary tissue of large yellow croaker(Larimichthys crocea)and obtain monolayer gonadal primary cells,the primary cells from ovary and testis of large yellow croaker were cultured by direct adherent method and subcultured by 0.25%trypsin digestion.Cells were cultured in DMEM/F-12(HAM)1∶1 with 20%FBS,100 IU/mL Penicillin and 100μg/mL Streptomycin at 26℃.Results showed that fibroblasts began to migrate form the ovary tissue of large yellow croaker on the 4th day after adherent.Epithelial cells migrated out and grew‘stone-like’on the 6th day after adherent.Different degrees of adherent inhibition were observed around the 17th day after adherent.Fibroblasts began to migrate form the testis tissue of large yellow croaker and on the 5th day after adherent.During the 9th day to the 17th day after adherent,some fibroblasts grew like vortex with long spindle shape and some were distributed evenly in triangular shape.Some cells cavitated seriously after culture to about the 18th day after adherent.The primary cells of ovary and testis tissue were digested and subcultured by trypsin on the 8th day after adherent.On the second day after digestion,some cells attached to the wall divided and proliferated.The second passage could be carried out on the 3th day after digestion.After the second passage,the growth of cells slowed down and the vacuolization of cells accelerated.