饲养层条件下昆明小鼠胰岛来源胰腺干细胞的建系及鉴定
Establishment and identification of pancreatic stem cell strain derived from islets of Kunming mice under feeder layer conditions
摘要 背景:到目前为止关于胰腺干细胞能建株甚至建系的报道极少,而且建系率较低,纯化出胰腺干细胞仍有一定的困难.目的:探索一套更合理、更有效的胰腺干细胞在体外分离及连续传代的技术和方法,使其有望能建立成细胞株甚至细胞系,为后续胰腺干细胞治疗糖尿病的相关研究奠定基础.方法:实验首先通过Percol不连续密度梯度离心的方法,使小鼠胰腺的内、外分泌部的细胞分布在不同的密度界面,然后获得来源于胰腺内分泌部即胰岛的干细胞,使用经丝裂霉素C作用的小鼠胚胎成纤维细胞作为饲养层,在饲养层条件下对来源于胰岛的胰腺干细胞进行连续传代,使其连续传至第30代,并通过生长特性检测、形态学观察、相关分子标志物鉴定以及分化特性鉴定等一系列的检测完善对该胰岛来源的胰腺干细胞株检测和鉴定.结果与结论:在连续传代过程中胰腺干细胞均体现了活跃的分裂增殖能力,并保持典型的干细胞形态学特征、表达胰腺干细胞分子标志物Nestin,经诱导分化后表达胰岛素基因,体现出了分化潜能.结果说明,实验饲养层条件下成功建立并鉴定了昆明小鼠胰岛来源的胰腺干细胞株.
Abstract:
BACKGROUND:Until now, little has been reported on establishment of pancreatic stem cell strains and lines,and the purification of pancreatic stem cells is difficult since the cell line establish rate is low.OBJECTIVE:To explore a more rational and effective technique of in vitro separation and continuous passage of pancreatic stem cells, with the hope to establish cell strains and even cell lines and to lay the foundation for the follow-up study of pancreatic stem cells in the treatment of diabetes.METHODS:Firstly, Percoll discontinuous density gradient centrifugation method was applied to separate the mouse pancreatic endocrine portion from the exocrine portion, then to obtain cell strains with highly proliferative ability and low differentiation from pancreatic endocrine portion-the islet. We used mouse embryonic fibroblasts treated with mitomycin C as a feeder layer, for in vitro continuous culture of islet-derived pancreatic stem cells under feeder layer conditions until they were transferred to the 30th passage to establish cell lines. Then pancreatic stem cell line derived from pancreatic islet was detected and identified by a series of tests including growth characteristic test, morphological observation, related molecular marker identification and differentiation characteristic identification.RESULTS AND CONCLUSION:In the continuous process of passage, pancreatic stem cells showed active proliferative ability, and maintained the typical morphological characteristics of stem cells and expression of pancreatic stem cell marker-Nestin. After induction, pancreatic stem cells showed insulin gene expression,reflecting their differentiation potential. Therefore, under the condition of feeder layer, the pancreatic stem cell line derived from Kunming mice was successfully established and the related identification was completed,which lays the foundation for the following research.
Author: Cen Yan-hui Yang Rui Jia Wei Li Zhong-hua Zhong Zhen-guo Zhong Jing Bao Juan He Guo-zhen Wu Xiao-jun Li Yi-yi
作者单位: 广西中医药大学基础医学院,广西壮族自治区南宁市 530222;广西中医药大学第一附属医院,广西壮族自治区南宁市 530023 广西中医药大学基础医学院,广西壮族自治区南宁市,530222
刊 名 中国组织工程研究 ISTICPKU
年,卷(期): 2017, 21(13)
分类号: R394.2
在线出版日期: 2017年6月12日
基金项目: 国家自然科学基金项目,广西高校科研课题,广西高校青年教师能力提升计划,广西壮族自治区卫生厅中医药科技专项课题,广西中医药大学学生科研训练课题,the National Natural Science Foundation of China, No. 81503406,University Research Projects in Guangxi, No. ZD2014068,Project of Improving the Basic Ability of Young Teachers in Universities in Guangxi,No. KY2016YB218, KY2016YB224,Special Scientific Project of Traditional Chinese Medicine supported by Health Department of Guangxi Zhuang Autonomous Region, No. GZPT13-04, GZBZ16-07, GZLC16-23,Student Research Training Project of Guangxi Medical University, No. 2014DXS02