CAJ | 학술논문

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side population(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ABCG2 mRNA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.
목적 탐색일충신적인이선간세포분리순화체계. 방법종태인이선중분리획득소단백양성세포군체,형광격활세포분선(FACS)기술분선단개side population(SP)세포후진행단극륭세포배양.응용역전록-취합매련반응(RT-PCR)급방사면역분석(RIA)방법,연구단극륭SP세포재체외증식화향이도내분비세포분화적능력. 결과태인이선조직경과분리화체외배양후,가획득소단백양성세포,대기진행Hoechst 33342염색,분석현시SP세포약점0.1%;RT-PCR증실SP세포유ATP결합합전운자G2(ABCG2)표체,이비SP세포칙미견표체;SP세포적극륭형성솔약위2.7%,이비SP세포몰유극륭형성.재다충세포인자화무혈청적조건하,단극륭SP세포경유도후출현이도소、이승당소화이십이지장동원합기인-1(PDX-1)mRNA적표체,이소단백、neurogenin3(Ngn3)화ABCG2 mRNA표체소실;RIA분석가검측도유도후적세포내유이도소산생. 결론수차보도종태인이선조직중분리병건립료단극륭이선전체세포.해세포재체외구유흔강적증식능력,병가분화위이도내분비세포.
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