单克隆人胰腺干细胞分化特性的研究

研究1例来源于4月龄男性流产胎儿胰腺组织的单克隆人胰腺干细胞（monoclonal human pancreatic stem cell，mhPSC）系的体内外分化特性。将mhPSCs接种在铺有0．1％明胶的培养皿内，扩增培养3d后，加高糖DMEM诱导液诱导培养25d。相差显微镜下．观察细胞生长状况。采用双硫腙染色法、RT—PCR及葡萄糖刺激释放胰岛素和C肽实验．对体外定向诱导mhPSCs分化为功能性胰岛进行检测。将mhPSCs悬液注射在成年雄性裸鼠腹股沟皮下．注射30d时，取出移植物，采用SP法进行免疫组织化学反应，以检测mhPSCs的体内自然分化潜能。体外扩增培养，mhPSCs贴壁生长，呈多角形上皮样。生长至单层．呈“铺路石”状。体外定向诱导，细胞逐渐由多角形变成圆形，并聚集成类胰岛。诱导培养15d时．形成的类胰岛中少数细胞分化为B细胞，双硫腙染色阳性。诱导培养25d时，多数细胞分化为8细胞，双硫腙染色阳性，转录表达胰岛素的mRNA。用不同浓度葡萄糖刺激．诱导胰岛不仅释放胰岛素和C肽，而且其释放量随糖刺激浓度升高显著增加（0．01〈P〈0．05）。体内分化实验显示，mhPSCs在裸鼠背部形成类畸胎瘤。类畸胎瘤易与裸鼠分离，色白，血管丰富。显著表达pdx1、胰岛素、胰高血糖素、CK、MBP及NF蛋白。该研究结果证实单克隆人胰腺干细胞系体外定向诱导分化为包含大量β细胞的功能性类胰岛，在体内自然分化为胰岛、上皮及神经组织细胞。

RESEARCH ON DIFFERENTIATION FEATURES OF THE MONOCLONAL HUMAN PANCREATIC STEM CELL

The in vitro and in vivo differentiation features of the monoclonal human pancreatic stem cell(mhPSC)line derived from the pancreatic tissues of a male abortive fetus at 4 month- old were studied.The mhPSCs were plated in culture dishes that had been coated with 0.1% gelatin in phosphate-buffered saline without calcium and magnesium.After proliferated for 3 days, the mhPSCs were induced in modified high-glucose Dulbecco's Modified Eagles's Medium for 25 days.The changes of the cell morphology were observed ...