从胎儿胰腺组织建立单克隆胰腺前体细胞的研究  

Monoclonal pancreatic progenitor cells established from human fetal pancreas

作　　者：张玲[1] 洪天配[1] 胡江[2] 刘羿男[2] 吴永华[1] 李凌松[2] 

机构地区：[1]北京大学第三医院内分泌科,100083 [2]北京大学干细胞研究中心

出　　处：《中华糖尿病杂志（1006-6187）》2004年第6期442-445,共4页

基　　金：国家自然科学基金资助项目 (3 0 170 44 3 ) ;国家 973重点基础研究发展规划基金资助项目 (2 0 0 1CB5 10 10 5 )

摘　　要：目的　探索一种新的人胰腺干细胞分离纯化体系。　方法　从胎儿胰腺中分离获得巢蛋白阳性细胞群体 ,荧光激活细胞分选 (FACS)技术分选单个sidepopulation(SP)细胞后进行单克隆细胞培养。应用逆转录 聚合酶链反应 (RT PCR)及放射免疫分析 (RIA)方法 ,研究单克隆SP细胞在体外增殖和向胰岛内分泌细胞分化的能力。　结果　胎儿胰腺组织经过分离和体外培养后 ,可获得巢蛋白阳性细胞 ,对其进行Hoechst 33342染色 ,分析显示SP细胞约占 0 1% ;RT PCR证实SP细胞有ATP结合盒转运子G2 (ABCG2 )表达 ,而非SP细胞则未见表达 ;SP细胞的克隆形成率约为 2 7% ,而非SP细胞没有克隆形成。在多种细胞因子和无血清的条件下 ,单克隆SP细胞经诱导后出现胰岛素、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、neurogenin3(Ngn3)和ABCG2mRNA表达消失 ;RIA分析可检测到诱导后的细胞内有胰岛素产生。　结论　首次报道从胎儿胰腺组织中分离并建立了单克隆胰腺前体细胞。该细胞在体外具有很强的增殖能力 ,并可分化为胰岛内分泌细胞。Objective To explore the possibility for establishing monoclonal pancreatic progenitor cells from the nestin positive cells of human fetal pancreas Methods Nestin positive cells were isolated from human fetal pancreas Single side population (SP) cell was sorted from the nestin positive cells via fluorescence activated cell sorting (FACS) and cultured for the assay of clone formation The capability of monoclonal SP cells to proliferate and differentiate into pancreatic islet endocrine cells in vitro were further studied by using reverse transcription polymerase chain reaction (RT PCR) and radioimmunoassay (RIA) Results Nestin positive cells were obtained from human fetal pancreas via cell culture in vitro The SP cells accounted for about 0 1% in the nestin positive cells via FACS analysis after Hoechst 33342 staining RT PCR revealed that ATP binding cassette transporter G2 (ABCG2) mRNA was expressed in the sorted SP cells, but not in the non SP cells The rate of clone formation was about 2 7% for the SP cells, whereas there was no clone formation for the non SP cells 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, neurogenin 3 (Ngn3) and ABCG2 disappeared in the monoclonal SP cells exposed to serum free media with the cocktail of several growth factors Furthermore, the intra cellular insulin content was detected by RIA in the SP cells after the induction Conclusion It has been shown for the first time that the monoclonal pancreatic progenitor cells are isolated from human fetal pancreas and that they have highly proliferative potential and the capability to differentiate into insulin producing cells in vitro

关 键 词：单克隆 前体细胞 胰腺组织 胎儿 表达 巢蛋白阳性细胞 SP RIA 胰岛素 RT-PCR 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]