大鼠胰腺导管上皮细胞体外分离与定向分化  

Isolation and differentiation of pancreatic ductai epithelial cells of rats in vitro

作　　者：刘涛[1] 勾善淼[1] 吴河水[1] 熊炯炘[1] 周峰[1] 万赤丹[1] 王春友[1] 

机构地区：[1]华中科技大学同济医学院附属协和医院胰腺外科,武汉430022

出　　处：《中华器官移植杂志》2007年第8期496-499,共4页Chinese Journal of Organ Transplantation

基　　金：国家自然科学基金(30170911)

摘　　要：目的分离和纯化大鼠的胰腺导管上皮细胞,在体外培养并诱导其向胰岛细胞定向分化。方法采用胶原酶逆行灌注法消化、密度梯度离心结合不同细胞贴壁差异性分离和纯化胰腺导管上皮细胞;以角蛋白-19(CK-19)免疫细胞化学染色进行鉴定;用RMPI 1640+含体积分数为10%的胎牛血清(FBS)培养基培养促进胰导管上皮细胞增殖,1周后,更换无血清培养基DMEM/F12并加入角朊细胞生长因子等进一步促进其增殖,细胞达80%汇合时传代,加入高糖及尼克酰胺促进胰导管上皮细胞向胰岛细胞定向分化;对胰岛样结构行双硫腙染色。结果CK-19染色结果证实所获细胞绝大多为导管上皮细胞。体外培养中导管上皮细胞24h开始贴壁,14～21d达80%融合并形成细胞克隆,第28d胰岛细胞样结构形成,且被双硫腙染成猩红色。结论采用密度梯度离心结合差异贴壁法可获得纯化的大鼠胰腺导管上皮细胞,在体外培养与诱导分化条件下可生成胰岛样结构。Objective To isolate and purify pancreatic ductal epithelial cells in adult rats, and induce differentiation of pancreatic ductal epithelial cells to islets in vitro. Methods By retrograde injection of collagenase into biliary-pancreatic tract, pancreatic tissues were digested and different types of pancreatic cells including islets, duct and degranulated aicni cells were separated by means of density gradient centrifugation. Ductal cells were purified by adhering method and identified by immunocytochemistry stain of ductal epithelial cells maker antigen （Cytokeratin 19, CK-19）. Ductal cells were expanded in RMPI 1640 with 10% FBS. About one week when most adherent cells were of monolayer, the medium was changed to serum-free DMEM/F12 supplemented with keratinocyte growth factor （KGF） to further expand ductal epithelial cells. When cells reached 80 % confluence, nicotinamide and high concentration of glucose were added to promote differentiation of pancreatic ductal epithelial cells. Islets-like-structure was stained by Dithizone. Results Immunocytochemistry stain of CK-19 revealed that most isolated cells were ductal epithelial cells. The cultured ductal epithelial cells began to adhere at day 1, reached 80 % confluence and cell clones were formed at day 14-21. At day 28, isletslike-structure appeared and was positive for Dithizone staining. Conclusions Ductal epithelial cells of rats can be isolated by means of density gradient centrifugation and purified by adhering method. Ductal epithelial cells can differentiate into islets-like-structure in vitro.

关 键 词：大鼠 胰腺 上皮细胞 细胞分化 胰岛 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]