体外培养山羊成纤维细胞系方法的建立

A method established for goat fibroblast in vitro

用山羊组织块和0．25％的胰蛋白酶消化培养法获得正常传代细胞，对于出现的上皮样细胞和成纤维样细胞混合生长的问题，则根据两者贴壁紧实程度不同，用0．05％的胰酶-EDTA进行不同时间的消化，将其分离纯化。纯化的成纤维体细胞经数次传代培养后，进行冷冻一解冻检验表明仍具有正常的传代能力。各代体细胞的核型分析表明，在体外培养至20～30代成纤维细胞的细胞形态（为梭形细胞，高度汇合后呈火焰状）、细胞周期以及核型均为正常，符合体细胞克隆转基因的基本要求。

Goat fibroblasts were cultured by tissue pieces, trypsin, respectively. Establish an ideal cellular culture methods. Especially, in the tissue pieces, first of all, we cut the tissue into 1mm^3 pieces, second dry the pieces by the centrifugation, third, add a little fetal bovine serum onto pieces, and then, paved the piece onto the culture plate in even, cultured for 60-90 min. in incubator, last, add the culture medium, The two method had a 100% of primary passagey fibroblast cells present rate and successful passage rate, Two methods had a differences between primary fibroblast cells present rate and time of can be passaged. Using different digested time to purified fibroblast cells with epithelial cells, Purified fibroblast cells still have normal capacity of passage after freeze-thawed. Kary otype analyze and cell cycle indicate that after 20- 30 passage of in vitro culture no abnormal. It suit for the need of clone and transgenic chone.