新生昆明小鼠胰腺巢蛋白阳性细胞的自然分化  

Natural differentiation of pancreatic Nestin-positive cells in neonatal Kunming mice

作　　者：叶健 何少健[2] 刘琼东 陈维平[2] 莫似恩 袁路[2] 胡利霞[2] 

机构地区：[1]广西壮族自治区人口与计划生育研究中心,广西壮族自治区南宁市530021 [2]广西医科大学组织学与胚胎学教研室,广西壮族自治区南宁市530021

出　　处：《中国组织工程研究与临床康复》2009年第14期2680-2684,共5页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：广西科技厅资助项目(桂科青0229009;桂科攻0592007-1G;桂科自0832157);广西计划生育委员会科研课题(桂人口计生研0502)~~

摘　　要：背景:胰腺干细胞在常规培养条件下易于分化,难以获得足够数量的种子细胞。目的:探讨体外培养条件下小鼠胰腺内、外分泌部的巢蛋白阳性细胞的分化情况。设计、时间及地点:细胞学体外观察,于2006-08/2007-08在广西医科大学基础医学院中心实验室完成。材料:SPF级新生昆明系小鼠25只,由广西医科大学实验动物中心提供。方法:以Ⅳ型胶原酶消化小鼠胰腺组织,采用Percoll不连续密度梯度法分离单个细胞混悬液,分别从第1界面(磷酸盐缓冲液/1.068g/L Percoll液)、第2界面(1.068g/L Percoll液/1.096g/L Percoll液)、第3界面(1.096g/L Percoll液/1.118g/L Percoll液)收集细胞,添加角质细胞生长因子、碱性成纤维细胞生长因子的低糖DMEM培养基进行体外连续培养。主要观察指标:双硫腙染色判断各界面细胞的来源,免疫组织化学检测各界面细胞巢蛋白、胰十二指肠同源盒基因蛋白1、角蛋白19的表达。结果:从第1,2界面收集到的细胞80%～90%双硫腙染色后胞质呈铁红色,表明主要来源于胰腺的内分泌部;从第3界面收集到的细胞双硫腙染色后胞质不染色,表明来源于胰腺的外分泌部。从胰腺的内、外分泌部均可分离出大、圆、单个核的细胞,巢蛋白染色呈阳性表达。随着体外培养时间的延长,来源于胰腺内分泌部的巢蛋白阳性细胞形态保持不变,呈集落样生长,表达胰十二指肠同源盒基因蛋白1,有向胰腺内分泌部β-细胞分化的趋势;来源于胰腺外分泌部的巢蛋白阳性细胞呈铺路石样形态,表达细胞角蛋白19,出现向导管上皮细胞分化的趋势。结论:新生昆明小鼠胰腺的内分泌部和外分泌部均存在巢蛋白阳性的细胞,前者具有分化为β-细胞的能力,后者具有分化为导管上皮细胞的能力。BACKGROUND： Pancreatic stem cells can easily differentiate under conventional culture conditions, and it is difficult to obtain enough seed cells. OBJECTIVE： To explore differentiation of Nestin-positive cells in mouse pancreatic gland and exocrine region under in vitro culture. DESIGN, TIME AND SETFING： The cytology, in vitro, observation experiment was conducted at the Central Laboratory, Basic Medical College, Guangxi Medical University from August 2006 to August 2.007. MATERIALS： A total of 25 SPF neonatal Kunming mice were provided by Animal Experimental Center, Guangxi Medical University. METHODS： Mouse pancreatic tissue was digested using type Ⅳ collagenase. Single cell suspension was isolated utilizing the Percoll discontinuous density gradient centrifugation. Cells were harvested from the first layer （phosphate buffer saline/1.068 g/L Percoll solution）, the second layer （1.068 g/L Percoll solution/1.096 g/L Percoll solution） and the third layer （1.096 g/L Percoli solution/l.118 g/L Percoll solution）. Cells were incubated in low-glucose DMEM containing keratinocyte growth factor and basic fibroblast growth factor in vitro. MAIN OUTCOME MEASURES： Dithizone staining was used to assess the source of cells from various surfaces. Immunohistochemistry was utilized to detect Nestin, pancreatic duodenal homeobox-1 （PDX-1） and keratin-19 expression. RESULTS： 80% 90% cells from the first and second layers presented red cytoplasm following dithizone staining. This suggested that the source was pancreatic endocrine portion. Cells from the third layer were not stained by the dithizone staining. This indicated that the source was pancreatic exocrine portion. Big, round, single-nucleous cells could be isolated from the pancreatic endocrine and exocrine portions, and were positive for Nestin staining. With the prolonged culture time in vitro, Nestin-positive cells from the pancreatic endocrine portion maintained unchangeable morpheus, grew in colonies, expressed PDX-1, and had the tendency

关 键 词：胰腺 巢蛋白 分化 

分 类 号：R394.2[医药卫生—医学遗传学]