猪胰管上皮细胞体外诱导分化为胰岛的形态鉴定与功能测定  

In vitro cultivation and differentiation of porcine pancreatic duct epithelial cells and identification of morphology and function

作　　者：刘涛[1] 范骥[1] 许逸卿[1] 万赤丹[1] 周峰[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：《中华实验外科杂志》2004年第7期836-838,共3页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金资助项目(30170911)

摘　　要：目的对成年猪胰管上皮细胞体外培养,促进增殖与分化,并对衍生胰岛进行形态鉴定和功能测定。方法在有血清培养基中行导管上皮细胞原代培养,无血清培养基中加入表皮生长因子和尼克酰胺诱导胰管上皮细胞分化,衍生胰岛行双硫腙(DTZ)染色,逆转录多聚酶链反应(RTPCR)检测胰岛素基因(Ins)和上皮细胞标志抗原(CK19)在衍生胰岛和成熟胰岛中表达,放射免疫法(RIA)测定衍生胰岛与成熟胰岛分泌胰岛素的功能。结果培养第28天细胞汇合并开始出芽形成胰岛样细胞团;衍生胰岛双硫腙染色阳性,同时表达Ins基因和CK19基因,成熟胰岛仅表达Ins基因;衍生胰岛在基础相胰岛素分泌量为(1.86±1.24)ng,刺激相为(4.43±1.59)ng,差异有显著性(P<0.05)。结论猪胰管上皮细胞在体外培养条件下可分化为胰岛,衍生胰岛表达β细胞表面标志并能分泌胰岛素。Objective To culture and differentiate porcine pancreatic duct epithelial cells in vitro and identify the morphology and function of derived islets.Methods Epithelial cells were expanded in RMPI 1640 medium containing serum and differentiated in DMEM/F12 serum free media with supplement of epithelial growth factor and nicotinamide.Buds and derived islets were stained by dithizone;The expression of insulin and CK 19 gene was detected by RT PCR;Contents of insulin in vitro were measured by radiological immunological assay (RIA) after islets were exposed to glucose stimuli.Results Epithelial cells receached confluence and buds appeared on the day 28 and were dithizone positive staining.They simultaneously expressioned Ins and CK 19 gene while mature ones only expressioned Ins.Secreteing insulin of derived islets were (1.86±1.24) ng and (4.43±1.59) ng respectively with high glucose stimuli or not and there was significant difference ( P <0.05).Conclusion Adult porcine duct epithelial cells can differentiate into insulin producing cells in vitro.Derived islets are positive for expression of β cells marker and can secrete insulin.

关 键 词：猪胰管 上皮细胞 体外诱导分化 胰岛 形态鉴定 功能测定 胰岛移植 

分 类 号：R657.5[医药卫生—外科学]