中文摘要：

目的研究1例单克隆人胰腺干细胞系的细胞形态和表达特性。方法10例4～5月龄流产胎儿胰腺组织,胶原酶消化。原代细胞DMEM培养,胰蛋白酶消化传代。在传代中逐渐纯化胰腺干细胞。克隆环筛选单克隆人胰腺干细胞,扩增培养。活力较好的细胞液氮冷冻保存。采用HE和Giemsa染色,观察单克隆人胰腺干细胞的形态特征;通过透射电镜分析其超微结构。用免疫组织化学反应和RT-PCR方法,检测其蛋白水平和mRNA水平的表达特征。结果1例来源于4月龄男性胎儿的单克隆人胰腺干细胞建系,传50代。液氮保存细胞1×109个以上。单克隆人胰腺干细胞完全贴璧时呈多角形上皮样,单核,圆形或椭圆形,单、双或多核仁。细胞核质比大,线粒体、内质网等细胞器均不发达,胞质内无分泌颗粒,属于发育早期的胰腺上皮样细胞。共表达胰肠同源域因子1（pdx1）、胰高血糖素、巢蛋白及角蛋白19（CK19）,不表达胰岛素、周期蛋白34（CD34）、CD44及CD45。转录表达pdx1、胰高血糖素及巢蛋白的mRNA,不转录表达胰岛素。结论首次证实1例共表达pdx1、胰高血糖素、巢蛋白及CK19蛋白的单克隆人胰腺干细胞系。

英文摘要：

Objective Cell morphology and expression features of the monoclonal human pancreatic stem cell line were studied. Methods Pancreatic tissues were taken from ten abortive fetuses of 4-5 month-old by sterile procedures,and digested with collagenase. Primary pancreatic cells were cultured in Dulbecco＇s Modified Eagles＇s Medium. These cells were further digested with trypsin for propagation. Pancreatic stem cells were gradually purified during generations. The monoclonal human pancreatic stem cells were selected by clone-ring,and further proliferated. Cells which had better viability were preserved in liquid nitrogen. Morphological features of the monoclonal human pancreatic stem cells were observed by hematoxylin-eosin and giemsa staining. Ultrastructure was analyzed by transmission electron microscope. Expression characteristics were demonstrated by immunocytochemistry staining and reverse transcription-polymerase chain reaction （RT-PCR）. Results A monoclonal human pancreatic stem cell line which was derived from a male abortive fetus of 4 month-old was established,and had been passed though 50 generations. More than 1×109 monoclonal human pancreatic stem cells were cryo-preserved in liquid nitrogen. The monoclonal human pancreatic stem cell had a polygonal epithelioid morphology with a round or oval caryon and mono or double or poly-nucleolus. The ratio of caryon and cytoplasm was large. There were underdeveloped endoplasmic reticulum,mitochondria,and no excretory granule in cytoplasm. These cells were positive for the pancreatic and duodenal homeobox factor 1 （pdx1）,glucagon,nestin and cytokeratin 19 （CK19）,and negative for the insulin,cyclin-dependent 34 （CD34）,CD44 and CD45 protein expression. Also,transcription factors of the pdx1,glucagon and nestin were expressed. Conclusion A monoclonal human pancreatic stem cell line which co-expressed the pdx1,glucagon,nestin and CK19 proteins was established and identified for the first time.