胎儿胰腺组织中巢蛋白阳性细胞的分离和体外增殖以及诱导分化
The proliferation and differentiation of nestin-positive cells isolated from human fetal pancreas
摘要 目的研究从胎儿胰腺组织中分离巢蛋白(Nestin)阳性细胞以及该细胞的体外扩增与向胰岛内分泌细胞分化的能力. 方法采用胶原酶消化法,从胎儿胰腺组织中分离获得胰岛样细胞簇(islet-like cell clusters,ICCs), ICCs经手工挑拣后接种,待形成单层上皮样细胞后,进行传代培养和诱导分化.利用逆转录-聚合酶链反应(RT-PCR)、免疫荧光染色及放射免疫分析(RIA)等方法,检测该细胞中分子标志物的表达,并对其向胰岛内分泌细胞分化的能力进行鉴定. 结果 (1)上述单层上皮样细胞具有很强的增殖能力,可至少连续传16代;(2)RT-PCR、免疫荧光染色分析显示,该细胞可表达干细胞的标志分子巢蛋白和ABCG2;(3)RT-PCR分析显示,在多种细胞因子和无血清的条件下,巢蛋白阳性细胞经诱导后可出现胰岛素、胰升糖素和胰十二指肠同源盒基因-1(PDX-1)mRNA的表达,而巢蛋白和Neurogenin3(Ngn3)mRNA表达消失.RIA分析也可检测到诱导后的细胞内有胰岛素产生. 结论从胎儿胰腺中分离得到的巢蛋白阳性细胞具有胰腺前体细胞的特性,在体外具有很强的增殖能力,并可诱导分化为胰岛内分泌细胞.该细胞有望为胰岛移植提供一种新的细胞来源.
Abstract:
ObjectiveTo isolate nestin positive cells from human fetal pancreas and to detect their capability of proliferation and differe ntiation into pancreatic endocrine cells in vitro.MethodsThe islet like cell clusters (ICCs) were isolated from human fetal pancreas with c ollagenase digestion. The free floating ICCs were hand picked up and cultured in a new dish. After the ICCs developed into the monolayer epithelium like cell s, they were passaged and induced to differentiate. Reverse transcription polyme rase chain reaction (RT PCR), immunofluorescence stain and radioimmunoassay (RI A) were used to detect the expression of their molecular markers and to identify their capability of differentiating into pancreatic endocrine cells in vitro. Results (1) The monolayer epithelium like cells had high prol iferative potential and could be passaged more than 16 times in vitro. (2) RT P CR and immunofluorescence stain analysis showed that these cells expressed nesti n and ABCG2, two of the markers of stem cells. (3) RT PCR analysis showed that the mRNA expressions of insulin, glucagon and pancreatic duodenal homeobox gene 1 (PDX 1) were detected,whereas the expressions of nestin and neurogenin 3 ( Ngn3) disappeared in the nestin positive cells treated with several cytokines a nd without serum. Furthermore, the intra cellular insulin content was detected by RIA after the induction. ConclusionNestin positive cells isolated from the human fetal pancreas possess the characteristics of pancreati c progenitor cells, and have high proliferative potential and the capability of differentiation into insulin producing cells in virto. This holds some promise of a new cell source for islet transplantation.
作者单位: 100083,北京大学第三医院内分泌科 北京大学干细胞研究中心
刊 名 中国糖尿病杂志 ISTIC
年,卷(期): 2003, 11(6)
分类号: R587
Keywords Nes tin
机标分类号: R58 R73
在线出版日期: 2004年3月5日
基金项目: 国家自然科学基金,国家"211"工程建设项目,国家重点基础研究发展计划(973计划)