胰腺干细胞转分化为胰岛样细胞与天然胰岛细胞的基因差异性

Gene differences between pancreatic stem cell-differentiated islet-like cells and normal islets

背景:目前胰腺干细胞在体外诱导转分化效率和分化成熟度较低,哪些基因在转分化中起关键性调控作用尚不清楚。目的:通过基因表达谱芯片分析转分化的胰岛样细胞与天然胰岛细胞在成熟度上的基因差异,筛选对转分化起关键性调节作用的基因。设计、时间及地点:基因水平的对照实验,于2004-07/2006-04在暨南大学第二临床医学院(深圳市人民医院)临床医学研究中心实验室完成。材料:SD大鼠胰腺分离的胰腺干细胞转分化的胰岛样细胞团,SD大鼠分离的天然胰岛。方法:分离和消化SD大鼠胰腺,用差异性贴壁、低血清培养等方法纯化出大鼠胰腺导管上皮细胞,利用免疫组化方法鉴定CK-19和PDX-1。通过含有exendin-4、角质细胞生长因子、尼克酰胺等细胞因子和葡萄糖的培养基,使之转分化为胰岛样细胞团,应用双硫腙染色和胰岛素免疫荧光染色鉴定。提取转分化的胰岛样细胞和天然胰岛细胞RNA,利用大鼠22000位点寡核苷酸芯片进行杂交,筛选差异性表达的基因,并进一步采用反转录-聚合酶链反应验证13个相关基因的表达。主要观察指标:转分化的胰岛样细胞团和天然胰岛鉴定,差异性基因筛选和鉴定。结果:筛选出差异表达基因1072个,其中表达上调的基因397个,上调10倍以上的基因35个;表达下调的基因有675个,其中下调20倍以上的基因37个。上调10倍以上的差异性基因中与细胞组织和生物发生相关基因最多,下调20倍以下的基因中与发育相关基因最多。对选定的13个与胰岛细胞发育密切相关的基因PDX-1,PDX-4,PDX-6,Nkx2.2,Nkx6.1,Nkx6.2,Ptfla,Isll,Ngn3,Myt1,Ptf1a,capn5,Ppy,通过反转录-聚合酶链反应进一步验证与芯片检测结果一致。结论:转分化的胰岛样细胞与天然胰岛细胞基因表达差异显著,参与胰岛发育的关键基因表达明显下调。

BACKGROUND： The efficiency and maturity of pancreatic stem cells is lower at present. Which gene plays an important role is not clear in differentiation. OBJECTIVE： To compare differential gene expression between pancreatic islet cells and natural islet using cDNA microarrays, and to screen out genes that play important roles in transdifferentiation. DESIGN, TIME AND SETTING： The gene controlled study was performed at Clinical Medical Research Center, Second Clinical Medical College （Shenzhen People＇s Hospital）, Ji＇nan University from July 2004 to April 2006. MATERIALS： Pancreatic ductal epithelial ceils were isolated from Sprague Dawley rat pancreatic tissue differentiated islet-like clusters and natural islet. METHODS： Pancreatic ductal epithelial cells were isolated from Sprague Dawley rat pancreatic tissue using differential adherence and low-dose serum, and then were identified with CK-19 and PDX-1 by immunocytochemical methods. Those cells were tansdifferentiated into islet-like clusters in glucose meduim containing exendin-4, keratinocyte growth factor and nicotinamide, and then identified by dithizone staining and insulin immunofluorescence. After isolating normal islets from rats, RNA was extracted separately from islet-like clusters and normal islets. Gene difference expression between islet-like clusters and normal islets were detected by using rat 22000 cDNA microarrays. According to the gene chip results, 13 differentially expressed genes were selected for further analysis by reverse transcription-polymerase chain reaction. MAIN OUTCOME MEASURES： The tansdifferentiated islet-like clusters and the normal islets were identified. Differentially expressed genes were screened and identified. RESULTS： 1 027 differentially expressed genes were screened, including 397 up-regulated genes （35 genes of ten-fold up-regulation） and 675 down-regulated genes （37 genes of 20-fold down-regulation）. Differential genes of over 10-fold up-regulation were mostly associated with cell tissues and biogenesis. Genes of 20-fold down-regulation were mostly correlated to development. The 13 genes associated with cell development （PDX-1, PDX-4, PDX-6, Nkx2.2, Nkx6.1, Nkx6.2, Ptfla, Isll, Ngn3, Mytl, Ptfla, capn5, Ppy） following reverse trauscription-polymerase chain reaction had identical results from microarray analysis. CONCLUSION： There are significant differences in gene expression between islet-like cells following transdifferentiation and normal islets. Expression of genes that participate in islet development is significantly down-regulated.