红鳍东方鲀精原干细胞鉴定、分离及纯化

IDENTIFICATION, SEPARATION AND ENRICHMENT OF SPERMATOGONIAL STEM CELLS FROM TIGER PUFFER(TAKIFUGU RUBRIPES)

为获得高比例精原干细胞,开展了不同月龄红鳍东方鲀精原干细胞分离纯化研究。采用组合酶消化法制备不同月龄的精巢单细胞悬液,通过形态学观察和生殖细胞特异基因vasa免疫荧光鉴别精原干细胞,Percoll不连续梯度(10%、30%和50%)纯化精原干细胞。结果显示: 14月龄雄鱼精巢生殖细胞主要为A型精原细胞,精原干细胞占比极显著性地高于22月龄(P<0.05)。纯化后精原干细胞主要分布在10%—30%Percoll梯度带中,14月龄雄鱼生殖细胞主要分布在此层,且纯化后精原干细胞占比远高于纯化前以及22月龄纯化前后。结果表明,14月龄更适用组合酶消化分离,并通过Percoll梯度离心法获得高比例的红鳍东方鲀精原干细胞。

This study investigated the isolation and purification of spermatogonial stem cells (SSCs) by single cell suspensions after enzyme digestion using testes of various months old tiger puffer.SSCs were enriched by Percoll discontinuous gradient.The SSCs were identified by optical observation and immunolocalization of Vasa protein.The majority of SSCs at 14-month-old fish was type A spermatagonial cells that were significantly higher than those of the 22- month-old males (P<0.05).SSCs were mainly enriched in 10%-30% Percoll gradient belt,where the germ cells were mainly distributed at 14-month-old fish.These results indicate that 14-month-old male fish is a great source for SSCs using the current isolation method.