牛成纤维细胞的分离与体外培养

研究了牛胎儿和成年牛皮肤组织成纤维细胞的分离、培养、纯化方法和生长特征。通过组织块贴壁培养和分离单细胞接种培养均能获得原代牛皮肤细胞。用2.5 g/L胰蛋白酶+1mmol/L EDTA和5 g/L胶原酶I联合消化牛皮肤组织较2.5 g/L胰蛋白酶+1 mmol/L EDTA消化,得到更多的单个细胞,两者之间差异极显著(P<0.01),但其死细胞比率却有较大升高;2.5 g/L胰蛋白酶+1 mmol/L EDTA消化牛胎儿组织得到的单细胞数显著高于皮肤组织消化后得到的细胞数(P<0.01),死细胞比率也高于同种酶消化的皮肤组织。分离纯化的胎儿和皮肤成纤维细胞的生长曲线都正常且相似。2.5 g/L胰蛋白酶+1 mmol/L EDTA消化贴壁细胞后死细胞率明显高于用0.5g/L胰蛋白酶+0.53 mmol/L EDTA消化的细胞(P<0.05);培养24 h后细胞贴壁率前者要明显低于后者(P<0.05)。用0.5 g/L胰蛋白酶轻度消化混杂生长的成纤维细胞和上皮样细胞,经过反复贴壁传代2～3代,可得到较纯的成纤维细胞。

The Dissociation and Culture of Bovine Fibroblasts in Vitro

The experiment were studied on the dissociation, culture and purification of bovine fetusfibroblasts and skin fibroblasts. The attachment and monolayer cells could be obtained by primary culture withbovine fetus and skin tissue. More fibroblast cells were obtained by 2.5 g/L trypsin+1 mmol/L EDTA and 5 g/Lcollagenase I digestion bovine skin tisssue than by 2.5 g/L trypsin+1 mmol/L EDTA (P<0.01), but digestion fetustissue by 2.5 g/L trypsin+1 mmol/L EDTA got a higher yield of cells than that of skin tissue (P<0.01). The growthcurves of fetus and skin fibroblast are normal and similarity, although the death rate of cells increased with thefibroblast yield increase. The death rate of attachment cells digested by 2.5 g/L trypsin+1 mmol/L EDTA wassignificantly higher than that by 0.5 g/L trypsin+0.53 mmol/L EDTA (P<0.05), and the attachment rate of the formerwas significantly lower than the latter (P<0.05) with 24 h culture. Homogeneous fibroblasts could be obtained by 0.5 g/Ltrypsin digestion of fibroblasts with small part of epithelial cells in it and by passed 2-3 subcultures.