【作者】 冯若鹏；

【导师】 窦忠英；

【作者基本信息】 西北农林科技大学 ， 临床兽医学， 2006， 硕士

【摘要】 糖尿病是危害人类健康的重大疾病之一,而传统的药物及胰岛素替代疗法却不能从根本上医治该病。近年来,随着免疫抑制剂的改进及细胞移植技术的不断成熟,移植胰岛细胞已成为治愈Ⅰ型和部分Ⅱ型糖尿病的有效途径。但是,由于每个临床胰岛移植受体通常需要2～4个新鲜尸体胰腺,胰岛供体短缺的矛盾亦更为突出,制约了利用胰岛细胞治疗糖尿病技术的应用。为进行胰岛移植,人们将移植的替代品转向异种动物-猪。猪繁殖性能强,组织来源充足,胰岛素结构和生物活性类似于人胰岛素,血糖调定点同人接近,猪胰腺细胞可在人血清中生存对人体无害,因此,胎猪胰腺干细胞可作为最佳的异种动物胰腺干细胞的供体细胞,对胎猪胰腺干细胞的研究是糖尿病治疗的新希望。本试验从胎猪胰腺不同部位分离得到干细胞并进行培养,生物学性能测定及诱导分化多能性等方面进行研究,为胎猪胰腺干细胞的临床应用提供基础资料。1在本试验室先前的研究基础上,筛选出适合胎猪胰腺导管源胰腺干细胞贴壁、培养的基础培养基为M199,生长所需的细胞外基质为明胶。以分次消化分次贴壁的方法纯化出胎猪胰腺导管源胰腺干细胞,该细胞表达CK-19、PDX-1阳性。研究中还发现部分原代胰腺干细胞表达胚胎干细胞的标志OCT-4,但这些细胞在传代培养过程中消失。分离纯化的胎猪胰腺导管源胰腺干细胞体外培养条件下增殖能力很强,现已传至18代。经含Nic、HGF的培养基诱导后可分化为胰腺内分泌细胞并分泌胰岛素,胰岛素释放量在第12～15d达到最大,证明诱导细胞在12-15d成熟程度达到最高,增长诱导时间只会加剧分化的内分泌细胞死亡。胎猪胰腺导管源胰腺干细胞经诱导后未能分化为心肌、神经、成骨细胞,说明该细胞不具有横向分化的多潜能性或采用的诱导方法不合适。2采用Zulewski等的方法从胎猪胰腺分离得到胎猪胰岛源胰腺干细胞,该细胞呈长梭形或成纤维样,生长时有聚集现象发生,可形成胚胎干细胞样集落。免疫组化鉴定该细胞表达CK-19、PDX-1、α-actin而不表达nestin。流式细胞仪检测细胞表面标志发现该细胞表达骨髓间充质干细胞的标志CD29、CD44、CD45、CD166说明该细胞可能具有和骨髓间充质细胞相类似的生物学特征。该细胞在体外具有较强的增殖能力第5、10、15代生长趋势相同。该细胞经诱导后可分化为胰腺内分泌细胞并释放胰岛素,不同于导管源胰腺干细胞的是该细胞在诱导第6d即可达到较好的分化效果。向成骨细胞、神经细胞分化试验的成功进一步说明本试验中分离到的胎猪胰岛源胰腺干细胞是一种存在于胰腺当中具有横向分化潜能的亚全能干细胞。更多还原

【Abstract】 Diabetes mellitus is a devastating disease suffered by about 150 million people in the world. At present, this disease could not cured completely by the therapy of traditional drugs or exogenous insulin injection. Along with the transplantation success of pancreatic islets and the improvement of immunosuppressive regimen, the possibilities of curing the type I and some of type II diabetes become more and more promising. However, the widespread application of this transplant technology has greatly been restricted by the deficient human pancreatic islets from the cadavers.In order to exert islet transplantation people turn their view to heterogeneity-porcine.The best xenogenic-animal surrogate pancreatic stem cells is the pancreatic stem cells of fetal piglets, it has many advantages which possess of unlimited donor-tissue source because high breeding, similar structure and bioactive with human being, approximative blood-glucose regulation-point with anthropobiology, surviving in human serum harmLess to body, therefore, the study on pancreatic stem cells of fetal piglets is becoming the hopeful cure of diabetes. This dissertation focuses on the study of fetal porcine pancreatic stem cells from which are the isolation and cultivation of fetal pancreatic stem cells,. the characterization of biology in pancreatic cells of fetal porcine , and the multipotents via inducing and differentiating. The basal data laid a foundation of clinical applications.1 This work based on the previous work in our laboratory,found that the most suitable medium and matrix for fetal porcine duct-derived pancreatic stem cell adhering and cultue was M199 and glatin.Purified the fetal porcine duct-derived pancreatic stem cell by the method of digesting and adhering,which was positive for CK-19 and PDX-1.In this study some of the primary culture cells were positive for the ESC marker OCT-4,but they lost in the process of subculture.The purified fetal porcine duct-derived pancreatic stem cells have good proliferate ability in vitro,when were induced by Nic and HGF these cells can differentiated into pancreatic endocrine cells and excrete insulin.The insulin secretion quantity reach the maximum in the 12-15d which proved that the cell reach the most mature stage,cells will die more if induced for more time.Fetal porcine duct-derived pancreatic stem cell cannot be induced to differentiate into smooth muscle、neuron or osteoblasts,which showed that these cell did not have the pluripotency or the method was not suitable for these cells.2 Adopt the method of Zulewski et al isolated the islet-derived fetal porcine pancreatic更多还原