大鼠胰腺干细胞的分离纯化、鉴定及定向分化
Separate purification and directed differentiation potency of pancreatic stem cells of adult rats in vitro
摘要 目的 探讨大鼠胰腺干细胞体外分离、纯化的实验条件及其定向分化潜能.方法 应用胰腺原位灌注V型胶原酶消化大鼠胰腺组织,100目滤网滤过,Ficoll 400浓度梯度离心法分离胰腺干细胞,采用添加表皮生长因子(EGF)、成纤维细胞生长因子(bFGF)的培养基行体外培养.运用荧光免疫染色法检测细胞表面CK-19、Pdx-1、Nestin、Insulin和Glucagon蛋白,计算阳性细胞率.双硫腙(DTZ)染色鉴定诱导分化后的细胞,光电酶标记法ELISA检测胰岛素分泌功能.结果 本实验获取的胰腺干细胞,体外培养可稳定传代培养8代以上.细胞表面CK-19、Pdx-1和Nestin蛋白均呈阳性,阳性细胞率分别为(88.6±6.2)%、(84.6±8.6)%和(81.3±7.5)%.诱导分化后细胞经DTZ染色呈棕红色.在高糖刺激下,培养上清液中胰岛素含量增高.结论本法可获取高纯度、多数量的胰腺干细胞,在人工诱导下可定向分化为具有胰岛素分泌功能的胰岛样细胞团,为临床胰岛移植获得足量细胞来源提供实验基础.
Abstract:
Objective To investigate the experimental condition of separate purification and directed differentiation potency of pancreatic stem cells of adult rats in vitro. Methods The pancreas of adult rats were digested by collagenase V in situ perfusion. Filtration was performed by 100 mesh filter. Ficoll 400 density gradient centrifugation was used to separate pancreatic stem cells and stem cells were for cultivation. Epidermal growth factor (EGF), fibroblast growth factor (bFGF) was added to medium for in vitro cultivation.Immunofluorescence staining method was used to detect the expression of CK19, Pdx-1, nestin, insulin and glucagon, and the positive cell rate was calculated. The differentiated cell was evaluated by dithizone (DTZ)staining; insulin excretion function was determined by optical enzyme labeled ELISA staining. Results The pancreatic stem cell obtained from the study could be cultivated in vitro for 8 generations. The expression of CK19, Pdx-1 and nestin of the tem cells were all positive, and the rate of positive cells were (88.6 ± 6.2)% ,(84.6 ±8.6)% and (81.3 ±7.5)%. The differentiated cell was in brownish red color by DTZ dye. After high concentration sugar stimulation, the expression of insulin secretion was increased in the supernatant.Conclusions This method can harvest highly purified, and large amount of pancreatic duct stem cell.Artificial induction may result directed differentiate for islet-like clusters and have insulin secrete function.
Author: SHI Guang-jun ZHANG Lei XU Ping YU Jiang
作者单位: 青岛市立医院肝胆咦外科,山东青岛,266071 济宁医学院附属医院急诊普外科
刊 名 中华胰腺病杂志 ISTIC
年,卷(期): 2010, 10(4)
分类号: R5
机标分类号: R68 TS2
在线出版日期: 2010年11月12日
基金项目: 青岛市科技局资金资助课题