人胰岛素原基因转染绵羊成纤维细胞及细胞株的建立

从动物耳皮肤组织采样 ,采用将组织块剪碎后直接贴附于培养瓶底部的方法进行原代培养 ,该方法使原代细胞出现率及可传代率均达到 10 0 %。根据上皮样细胞和成纤维样细胞贴壁紧实程度的不同 ,用 0 .0 5 %的胰蛋白酶-EDTA对其进行消化 ,可将两种不同类型的细胞进行分离和纯化。通过脂质体介导 ,以BLG -hINS(含乳球蛋白调控基因的人胰岛素原基因 )基因作为目的基因、GFP(绿色荧光蛋白 )基因作为标记基因共转染绵羊成纤维细胞 ,经G - 4 18筛选后 ,得到转染细胞。对转染的细胞分别用单细胞显微操作法和有限稀释法进行细胞克隆 ,两种方法均可得到克隆细胞。选形态正常、生长均匀的 5个细胞克隆进行PCR检测 ,结果 5个克隆均转有GFP基因 ,其中两个转有BLG -hINS基因。高代培养细胞、转染细胞和克隆细胞经核型分析后 ,染色体数目均为 2 7对 ,表明绵羊耳的成纤维细胞建立细胞株后 ,可以作为外源基因转染的有效供体细胞。

Transfection of Human Pro-insulin Gene into Sheep Fibroblast Cells and Establishment of the Transfected Cell Lines

Tissues from adult sheep ear skin were cultured and passaged using improved method of cutting into small pieces directly. We had a 100% of present for primary passage of fibroblast cells and successful passage rate for all the experimental adult sheep of different breed with this method. We had used 0.05% trypsine-EDTA at different digestion times to purified fibroblast cells from epithelial cells according to their capacity of adhering to the plastic bottom. BLG-hINS (β-lactoglobulin/human pro-insulin) gene was co-transfected with GFP (green fluorescent protein) gene as the selective marker using liposome to deliver DNA into target cells. After three weeks selecting by G-418, the stable transfected cells were obtained. These cells were subsequently cloned by following two ways: one is to passage the cells into 35 mm tissue culture dishes with very low cell density (about 50～100 cells/mL), and picked the individual healthy colonies into an other tissue culture dish after about a week culture; the other is to pick single cell into 96-well tissue culture dishes under microscope, allow them to expand into colonies. Healthy colonies were determined to assure the presence of both BLG-hINS and GFP genes by PCR. High passaged cells (adult sheep cells cultured up to 30 passages and sheep fetus cells cultured up to 45 passages), transfected cells and cloned cells have not been observed abnormal of chromosomes' number and ploidy by karyotyping analysis.