斑节对虾性腺组织、细胞的原代培养条件优化

通过观察不同成分培养基及不同培养方法培养的斑节对虾性腺组织及细胞原代培养生长情况,分析细胞活力及生长相关基因表达情况,判断3种改良培养基的适用条件,为进一步细胞培养试验提供支持。试验结果表明,以L-15培养基为基础培养基,不同配方的培养效果差距较大,其中机械分离法可成功培养离体斑节对虾性腺组织,而酶解法与3号培养基结合效果较好;在培养过程中可见离体卵母细胞多呈悬浮生长,部分可见分裂相,离体精原细胞多呈贴壁生长,1号、2号培养基中的细胞可维持良好生长状态达一周以上;CCK测试结果表明,机械分离的性腺组织和酶解法分离的性腺细胞分别在1号、3号培养基中有更高的细胞活力,而2号培养基中的性腺组织及细胞在两种培养方法下的活力差别不大。进一步的荧光定量PCR检测了离体培养过程中MAPK信号通路相关基因的表达变化情况,结果表明,Ras、c-Mos、MEK1、ERK基因在卵母细胞中表达变化更显著,且c-Mos基因与离体细胞生长发育情况呈正相关。本试验系统性地筛选了适宜斑节对虾性腺组织、细胞的原代培养方法,为了解培养过程中的生理过程变化提供了相关参考数据。

Optimization of Tissue and Primary Cell Culture of Tiger Shrimp Penaeus monodon

The growth,cell activity and related-genes expression level were investigated in gonad tissues separated by mechanics and enzymes and primary cell culture of tiger shrimp Penaeus monodon cultured in three improved medium using CCK method and qPCR to determine the suitable conditions of three kinds of improved culture media and to provide support for further cell culture test. The results showed that the gonad tissues of the tiger shrimp were successfully cultured in the media based on L-15 by the mechanical separation method,with different effects. The gonadal tissue of tiger shrimp was successfully cultured by mechanical separation method and in the third type of medium by the enzymatic hydrolysis method in vitro. The growth of oocytes in suspension was mainly observed,some in the division phase,and the spermatogonia were growth as adherent cells in the first and second types of media at a good growth state for more than a week. However,the CCK counting test revealed that there were higher cell activities in the first and third types of media by the two methods compared with the second one. The changes in MAPK signaling pathway were also observed by q PCR,indicating that the key factors of MAPK signal pathway (Ras,c-Mos,MEK1,ERK) in oocyte were significantly higher than those in spermatogonium cells,especially,the c-Mos gene being positively correlated with the growth and development of the cells in vitro. The findings provide relative reference for understanding of the physiological process changes in the culture process.