论文部分内容阅读
目的 完成胰腺导管上皮细胞的原代及传代培养 ,建立能够体外长期培养的胰腺导管上皮细胞系。 方法 用含有胶原酶 IA型的消化液消化分离胎儿胰腺组织为细小、均匀的细胞团进行接种 ,接种的细胞团用含10 %胎牛血清、4.0 m mol/L谷氨酰胺、0 .0 1%大豆胰酶抑制剂、0 .0 2 %牛血清白蛋白、5 .0 m g/L牛脑垂体提取物、2 .5× 10 - 3m g/L表皮生长因子、2 5 .0× 10 - 2 m g/L霍乱毒素、5 .0 mg/L胰岛素、10 .0 mg/L转铁蛋白、1.0 m g/L地塞米松、5 0 m g/L庆大霉素的完全培养基培养 2 4h后 ,换含 2 %胎牛血清的完全培养基继续培养 ,在细胞团达到 80 %～ 90 %的细胞汇合时 ,1∶ 2传代培养。 结果 获得的原代培养细胞不含有淀粉酶 ,表达细胞角蛋白 ,可初步认定为胰腺导管上皮细胞。原代培养的胰腺导管上皮细胞已传代培养到第 3代。 结论 我们的原代培养和传代培养的方法、条件适宜于胰腺导管上皮体外细胞培养。
Objective To establish primary and subculture of pancreatic ductal epithelial cells and to establish a pancreatic ductal epithelial cell line capable of long-term culture in vitro. Methods The fetal pancreas tissue was digested with collagenase type IA digestion to inoculate small and uniform cell mass. The inoculated cell mass was inoculated with 10% fetal bovine serum, 4.0 m mol / L glutamine, 0.2% bovine serum albumin, 5.0 mg / L bovine pituitary extract, 2.5 × 10 -3 mG / L epidermal growth factor, 25.0 × 10 - After incubation with 2 mg / L cholera toxin, 5.0 mg / L insulin, 10 mg / L transferrin, 1.0 mg / L dexamethasone and 50 mg / L gentamicin for 24 hours , With 2% fetal bovine serum complete medium continued to culture, in the cell mass reached 80% to 90% of the cell confluency, 1: 2 subculture. Results The primary cultured cells did not contain amylase, expressed cytokeratin, which could be initially identified as pancreatic ductal epithelial cells. Primary culture of pancreatic ductal epithelial cells have been subcultured to the third generation. Conclusion Our primary culture and subculture methods, conditions suitable for pancreatic duct epithelial cell culture in vitro.