广西巴马小型猪附睾成纤维细胞的分离培养和鉴定

Culture and Identification of Epididymis Fibroblast of Guangxi Bama Mini-Pigs

为了建立广西巴马小型猪附睾成纤维细胞体外培养体系,利用微量液体组织块贴壁法原代培养分离附睾成纤维细胞,进行常规传代培养、冷冻和复苏,并通过接触抑制和解除抑制研究细胞的增殖能力,以及通过免疫荧光对其进行鉴定。结果表明,该法成功分离获得广西巴马小型猪附睾成纤维细胞,并可以大量传代和冷冻,目前已经传至55代以上,生长良好;冷冻复苏和接触抑制解除后仍可以正常培养和传代;细胞免疫荧光检测,波形蛋白表达阳性。可见,通过微量液体组织块贴壁法能成功分离得到广西巴马小型猪附睾成纤维细胞,在体外能稳定培养和传代。

The objective of this study was to establish an in vitro culture system for epididymis fibroblast of Bama mini-pigs in order to provide new source of donor for the related fields of somatic cell cloning and transgene. Method： The primary culture epididymis fibroblast was isolated from the tissue mass by attachment method in a small amount of medium, and then the routine serial subcuhuring, freezing and recovery were conducted. The proliferation activity of the ceils were investigated through contact inhibition and removal of the inhibition, and the cells were identified through immunofluorescence detection. Result： The epididymis fibroblasts were isolated successfully, which could be applied for subeuhuring and freezing in a great quantity, and it had been subcultured for over 55 generations. It could still be cultured and subculturcd normally after the freezing-recover and removal of contact inhibition. The immunofluorcscence detection of the cells showed that the exprcssion of vimentin was positive. Conclusion ： The epididymis fibrcblast could be isolated successfully, which could be applied for stability in vitro culture and subculturing. It was an economic, practical, simple and stable method.