栉孔扇贝心脏细胞的体外培养  

Cell cultures from Chlamys farreri heart cells

作　　者：季爱昌 王华 晏萌[1] 张志峰[1] JI Aichang;WANG Hua;YAN Meng;ZHANG Zhifeng(Key Laboratory of Marine Genetics and Breeding,Ministry of Education;Ocean University of China,Qingdao 266003,China)

机构地区：[1]中国海洋大学海洋生物遗传育种教育部重点实验室,山东青岛266003

出　　处：《中国水产科学》2018年第2期346-353,共8页Journal of Fishery Sciences of China

基　　金：国家863计划项目(2012AA10A402)

摘　　要：体外培养细胞对研究生物的资源保护、功能基因及病害发生机理与防治等均具有重要意义。然而,目前海洋双壳贝类中可以长期存活的组织细胞是有限的。本研究采用植块法启动栉孔扇贝(Chlamys farreri)心脏细胞的原代培养,通过优化培养基的方法,建立了可使其长期存活的原代培养体系。根据组织块迁出细胞的数量和细胞存活时间,确定L-15是3个培养基(L-15,M199,L-15+M199)中最适宜栉孔扇贝心脏细胞的基础培养基。通过三因子三水平正交实验,得出栉孔扇贝心脏细胞适宜的添加物组合:L-15基础培养基中添加5%胎牛血清、50 mmol/L牛磺酸和6 mmol/L Ca^(2+)。原代培养产物中以心肌细胞为主要的细胞类型,其中部分心肌细胞在原代培养14 d内可进行节奏性搏动;部分细胞可局部形成心肌束和肌管样结构;心肌细胞在体外存活时间达2个月。本研究将为栉孔扇贝基础生物学和功能基因的研究提供细胞平台。Cells cultured in vitro play an important role in organism-resource protection and functional identification of genes,as well as mechanism exploration and prevention of animal disease.However,tissue cells that can survive for a long time in vitro are limited at present in marine shellfish.In this study,primary cultures of heart cells from the scallop Chlamys farreri were started using an explant method,and a primary culture system-which can keep the cells alive for a long time in vitro-was established using an optimizing-medium method.Effects of three basic media(L-15,M199,and L-15+M199)on the number of migrated cells from the explants and cell survival time were compared;the L-15 medium was verified to be the optimal basic medium for C.farreri heart cells.Furthermore,the optimal supplemental-factor combination for C.farreri heart cells was the L-15 medium supplemented with 5%FBS,50 mmol/L taurine and 6 mmol/L Ca2+(orthogonal experiment of three factors and three levels),in which cardiomyocytes could survive for 2 months in vitro.In this study,the results show that low concentration serum(5%FBS)was most favorable for the migration of cells from the tissue mass.With an increase in FBS concentration,the cell-migration ability was weaker,and the survival time of primary cells became shorter.After the addition of taurine,the survival time of primary cells was significantly prolonged.However,the Ca2+(2 mmol/L,4 mmol/L,and 6 mmol/L)had no significant effect on C.farreri heart-cell cultures.Perhaps the additive taurine regulated intracellular Ca2+to a suitable level,resulting in the addition of Ca2+without significant improvement in the primary culture.In this primary culture,most cells were cardiomyocytes;part of the cardiomyocytes beat at regular intervals within a short time,and cardiomyocytes and myotubes were formed in part of the region.This phenomenon was also reported in the primary culture of heart cells of Crassostrea gigas.This study provides a useful foundation for further studies on C.farreri basic biology

关 键 词：栉孔扇贝 心脏细胞 体外培养 正交实验 

分 类 号：Q751[生物学—分子生物学] S968[农业科学—水产养殖]