肝细胞生长因子及葡萄糖对人胰腺干细胞向胰岛素分泌细胞分化的影响

Influence of hepatocyte growth factor and glucose on differentiation of human pancreatic stem cell into insulin-producing cells

目的 探讨人胰腺干细胞体外扩增及其向胰岛素分泌细胞分化的影响因素.方法 应用剪碎消化法从孕4～6月龄引产的人胎儿胰腺中获得胰岛组织,选用差速贴壁法从胰岛中分离胰腺干细胞,经扩增后定向诱导成具有胰岛素分泌功能和胰岛样结构的胰岛样细胞团,观察肝细胞生长因子和葡萄糖对胰腺干细胞向胰岛素分泌细胞诱导分化以及胰岛样细胞团胰岛素分泌功能的影响.采用流式细胞仪和免疫组织化学法检测扩增前后nestin阳性细胞和ABCG2阳性细胞.行葡萄糖刺激的胰岛素释放试验,以酶联免疫吸附法（ELISA）检测每1×105个扩增细胞经定向诱导后形成的胰岛样细胞团释放的胰岛素量,比较肝细胞生长因子和葡萄糖对定向分化的影响.采用单因素方差分析或多样本均数两两比较进行统计学分析.结果 经过15代连续扩增,nestin阳性细胞和ABCG2阳性细胞分别扩增了27.9倍和37.8倍.扩增后的细胞经15 d定向诱导后形成具有胰岛素分泌功能和胰岛样结构的细胞团.葡萄糖刺激的胰岛素分泌试验显示,当诱导培养基中肝细胞生长因子浓度为0、5、10、15μg/L时,5.6 mmol/L葡萄糖刺激后,胰岛素分泌量分别为5.00、12.93、14.91和11.23μU;25.0 mmol/L葡萄糖刺激后,胰岛素分泌量分别为19.91、35.53、47.43和23.33 μU.诱导培养基中的葡萄糖浓度为0、2.8、5.6和11.2 mmol/L时,5.6 mmol/L葡萄糖刺激后,胰岛素分泌量分别为9.07、14.21、6.91和3.53 μU;25.0 mmol/L葡萄糖刺激后,胰岛素分泌量分别为26.64、48.97、37.63和34.20 μU.结论 人胰腺中nestin和ABCG2阳性胰腺干细胞具有很强的体外扩增能力.扩增后的胰腺干细胞仍然具有形成胰岛素分泌细胞的功能,经体外诱导后能形成胰岛样细胞团.在胰腺干细胞向胰岛素分泌细胞分化诱导过程中,适当浓度的肝细胞生长因子和葡萄糖有利于胰岛样细胞团的形成和胰岛素分泌.

Objective To investigate the influence factors of expansion and differentiation of human pancreatic stem cells into insulin-producing cells.Methods Human pancreatic stem cells were obtained from induced abortive fetal pancreatic islets.The pancreatic stem cells were expanded in defined inducingmedium for 15 passengers in vivo. The nestin-positive cells and ATP-binding-cassette-superfamily-G2（ABCG2） positive cells in expanded cells were detected by flow cytometer and immunohistochemistry.Expanded pancreatic stem cells were then induced to form islet-like cell clusters （ICCs）.Insulin secretion from ICCs was tested by glucose stimulatory insulin secretion （GSIS）.Influence of glucose and hepatocyte growth factor on insulin producing ability of ICCs was investigated during pancreatic stem cells differentiation into insulin-producing-cells.Insulin secretion from ICCs that differentiated from 1 × 105 expanded cells was used to estimate the efficiency of pancreatic stem cells differentiation.Results Nestin-positive cells and ABCG2-positive cells were expanded to 27.9 and 37.8 times when isolated pancreatic stem cells were cultured for 15 passengers.The expanded cells formed ICCs at 15 days in vitro.When hepatocyte growth factor concentration in culture medium was 0,5,10,and 15 μg/L,when stimulated with 5.6 mmol/Lglucose,insulin secretion from ICCs was 5.00,12.93,14.91 and 11.23μU,respectively. When stimulated with 25.0 mmol/L glucose,insulin secretion was 19.91,35.53,47.43 and 23.33μU,respectively.When glucose concentration in basic culture medium was 0,2.8,5.6 and 11.2 mmol/L,insulin secretion was 9.07,14.21,6.91 and 3.53μU,respectively,while stimulated with 5.6 mmol/Lglucose.While stimiulated with 25.0 mmol/L glucose,insulin secretion was 26.64,48.97,37.63 and 34.20μU. ConcIusion Nestin positive cells and ABCG2 positive cells in can be expanded in vitro.Expanded pancreatic stem cells could retain the ability of differentiation into insulin-producing cells and formation of ICCs. A proportionate hepatocyte growth factor and glucose concentration in inductive medium would be in favor of formation of ICCs and insulin secretion during differentiation of pancreatic stem cells into insulin-producing cells.