摘要
本研究首先将水牛睾丸经二步酶消化法制成单细胞悬液,依次采用差速贴壁法和Percoll不连续密度梯度离心法分离和纯化精原细胞。在制备好的小鼠胎儿成纤维细胞饲养层上,采用含2.5%血清的培养液对精原细胞进行体外培养,观察血清和成纤维细胞对精原细胞生长的影响,并在体外成功培养了2周。通过提取体外培养精原干细胞总RNA,设计引物并对其进行基因鉴定和免疫细胞化学鉴定,证实体外培养所得的细胞仍保持有精原干细胞的特性,并且该克隆是处在未分化状态的精原干细胞形成的。上述研究可为体外建立水牛精原干细胞长期培养体系提供技术支撑。
In this experiment, we used two-step enzymatic digestion to make buffalo testis into single cell suspension, and then purifid them with differential adhesion and percoll discontinueous density gradient centrifugation method. Spermatogonial cells were with mouse fetus fibroblast feeder layer and 2.5% FBS, and observed the influence of serum and fibroblasts on spenaatogonial cell growth in vitro, which showed successful culture in vitro for 2 weeks. By extracting total RNA of spermatogonial stem cells cultured in vitro, design primers and gene identification and immunocytochemistry, the formation of spermatogonial stem cells were confirmed in the undifferentiated state. The studies provide technical support for long-term in vitro culture of buffalo spermatogonial stem cells.
出处
《中国奶牛》
2013年第17期16-21,共6页
China Dairy Cattle
基金
国家现代农业(奶牛)产业技术体系建设专项资金(CARS-37)
中央高校基本科研业务费专项资金(2011PY020)共同资助
关键词
精原干细胞
体外培养
血清
饲养层
鉴定
Spermatogonial stem cells
In vitro culture
Serum
Feeder layer
Identification
