【摘要】 目的:培养小鼠胰腺导管上皮细胞并使之转分化为胰腺干细胞及胰岛样细胞,为胰岛移植治疗糖尿病提供细胞来源。方法:分离培养昆明小鼠胰腺导管上皮细胞,以添加有角化细胞生长因子(KGF)、肝细胞生长因子(HGF)和烟酰胺的DMEM/F12培养基培养,分别于光镜和电镜下观察细胞形态和超微结构的变化。采用细胞免疫化学染色检测第1天和16天时CK-19、PDX-1蛋白的表达,RT-PCR检测第1天和16天时胰岛素和胰高血糖素基因的表达,第21天行双硫腙染色试验及葡萄糖刺激的胰岛素释放试验。结果:培养第7~10d,细胞快速增殖成鹅卵石样;培养第16~21d,细胞逐渐聚集,最终形成胰岛样细胞团;培养第16 d,细胞超微结构的变化表明上皮细胞在向干细胞转分化。免疫化学染色显示,分离第1天,大部分细胞CK-19染色阳性,偶见PDX-1弱阳性细胞,第16天时,CK-19阳性细胞快速增殖形成细胞团,大部分细胞PDX-1染色阳性。RT-PCR显示培养第16天,细胞胰岛素和胰高血糖素表达较培养第1天明显增强(P<0.01)。培养第21天,胰岛样细胞团更加成熟,双硫腙着色阳性,且在高糖(15mmol/L)刺激下的胰岛素释放较低糖(5.6 mmol/L)刺激时增加了1.6倍(P<0.05)。结论:小鼠胰腺导管上皮细胞在体外培养条件下可增殖,并具有干细胞潜能,可转分化为胰岛素分泌细胞。更多还原

【Abstract】 Aim: To look for suitable cell-replacement source for treating DM,pancreatic ductal epithelial cells from adult Kunming mice were separated,cultured and transdifferentiated into islet-like cells.Methods:Pure pancreatic ductal epithelial cells were separated and cultured in DMEM/F12 medium with supplement of keratinocyte growth factor,hepatic growth factor and nicotinamide.Samples were taken at different time points for light and electron microscopy and for immunocytochemical study with antibodies against transdifferentiation gene PDX-1 and protein CK-19.The expressions of insulin and glucagon gene were detected by RT-PCR.DTZ staining experiment was done to examine whether differentiated islet-like clusters had endocrine function.Study was performed to detect whether the new β cells in these islet-like clusters could secrete insulin in response to glucose.Results: A large number of epithelial cells were CK-19 immunoreactive positive and few of them were PDX-1 positive at the beginning of isolation,then CK-19-positive cells proliferated quickly and formed substantial plaques of epithelial cells in cobblestone pattern after 7 days culture.Sixteen days later,these cells began to form islet-like clusters gradually,while most of them were PDX-1 immunoreactive positive.RT-PCR showed very low levels of insulin and glucagon mRNA in the beginning but increased as the process of differentiation.On the 21st day,islet-like clusters stained by DTZ revealed red.In those samples exposed to a stimulatory 15 mmol/L glucose,there was a 1.6-fold increase in insulin compared with those to 5.6 mmol/L glucose.Conclusion: Pancreatic ductal cells of adult Kunming mice could proliferate quickly and have the potency of transdifferentiating into islet-like clusters when cultured in vitro under appropriate conditions.更多还原