单克隆人胰腺干细胞分化特性的研究  

RESEARCH ON DIFFERENTIATION FEATURES OF THE MONOCLONAL HUMAN PANCREATIC STEM CELL

作　　者：效梅[1] 安立龙[1] 窦忠英[2] 

机构地区：[1]广东海洋大学农学院动物医学系,广东湛江524088 [2]西北农林科技大学国家干细胞工程技术中心陕西分中心,陕西杨凌712100

出　　处：《分子细胞生物学报》2008年第6期457-464,共8页Journal of Molecular Cell Biology

基　　金：国家高技术研究发展计划资助项目(863计划;No.2002AA216161);国家重点基础研究发展规划项目(973计划;No.G1999054301);国家自然科学基金资助项目(No.39970363);教育部重点资助项目(No.103160);陕西省重点攻关项目(2002k01-G3);广东省自然科学基金资助项目(No.04011471);广东省教育厅科学基金资助项目(No.2003-1009)~~

摘　　要：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 hu- man 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3 d后,加高糖DMEM诱导液诱导培养25 d。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30 d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15 d时。形成的类胰岛中少数细胞分化为β细胞,双硫腙染色阳性。诱导培养25 d时,多数细胞分化为β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。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 monthold 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 by phaseeontrast microscope during indueement course. The results of the mhPSCs in vitro induced to differentiate into functional pancre- atic islets were identified using dithizone staining, RT-PCR and stimulation-glucose secreting insulin and C-peptide radioimmunoassay. The mhPSCs suspension was separately injected under the groin hypoderm of male nude mice. On the 30^th day, the grafts were taken off. The immunochemistry reactions were performed by the SP method. The in vivo differentiation ability of the mhPSCs in nude mice was assessed. In vitro proliferation euhure, the mhPSCs adhesively grew and showed polygon epithelioid morphology. After proliferation a layer, the mhPSCs showed the gravelstone-like. During in vitro directional inducement, the mhPSCs gradually turned from polygon to round, suspended to grow and assembled pancreatic islets-like clusters. On the 15^th indueement day, only a few cells of pancreatic islet-like clusters were induced into the β cells that became crimson with dithizone staining. However, till the 25^th inducement day, most cells of pancreatic islet-like clusters had differentiated into the β cells, as identified by dithizone staining, which expressed transcription factor of insulin. Respectively stimulated with different concentration glucose, the induced pancreatic islets not only secreted insulin and C-peptide, but also the secretion volumes of the insulin and C-peptide were markedly increased after the stimulation with higher coneentration glucose （0.01〈P�

关 键 词：胰腺干细胞 单克隆 分化 人 

分 类 号：Q813[生物学—生物工程] Q785