成人脂肪源Flk1+CD31-CD34-细胞体外定向诱导分化为胰岛样细胞观察

目的:体外定向诱导成人脂肪源Flk1 CD31-CD34-细胞向胰岛样细胞分化.方法:首先将成人脂肪源Flk1 CD31-CD34-细胞培养在含适当浓度的B27、bFGF及EGF等因子的培养基(诱导液1)中诱导6 d,接着更换诱导培养基,用含适当浓度的β细胞调节素、尼克酰胺等高糖无血清培养基(诱导液2)诱导细胞向胰岛样细胞分化,诱导前后用RT-PCR法检测细胞nestin、神经元素3(ngn3)、胰岛素启动因子1(IPF-1)、胰岛素、胰高血糖素基因的表达;免疫组织化学法检测nestin、胰岛素、胰高血糖素、生长抑素抗原的表达;放射免疫分析法检测细胞胰岛素分泌情况.结果:成人脂肪源Flk1 CD31-CD34-细胞经诱导液1诱导6 d后可分化成nestin 的祖细胞;继续用诱导液2诱导6 d后变圆的细胞逐渐增多,并聚集成团,免疫荧光结果证实经2阶段诱导后的细胞表达胰岛素、胰高血糖素、生长抑素等内分泌激素,放射免疫分析结果表明诱导的胰岛样细胞团可以分泌胰岛素.结论:成人脂肪源Flk1 CD31-CD34-细胞具有胰腺干、祖细胞的固有特征,极有可能作为种子细胞用以糖尿病的细胞治疗.

Observation of differentiation of human adult adipose tissue-derived Flk1+CD31-CD34- cells into pancreatic islet-like endocrine cells in vitro

Aim:To promote differentiation of Flk1 CD31-CD34-cells isolated from human adult adipose tissue toward pancreatic islet endocrine cells in vitro.Methods:Flk1 CD31-CD34-cells were first cultured and plated in medium supplemented with B27,epidermal growth factor(EGF),and basic fibroblast growth factor(bFGF).Next,the glucose concentration in the medium was increased,bFGF was withdrawn,betacellulin and nicotinamide was added.RT-PCR immunofluorescence stain,fluorescence-activated cell sorting(FACS) and radioimmunoassay(RIA) were used to detect the expression of nestin,insulin,and other pancreatic endocrine hormones.Results:Initially,a nestin positive precursor cell population was found,then small round cells became more and more.Later on,they differentiated into islet-like clusters.The indueded cells resulted in the formation of clusters which exhibited higher insulin secretion and other pancreatic endocrine hormones.RT-PCR detected an enhanced expression of pancreatic genes in the differentiated cells.Immunofluorescence revealed a high percentage of insulin-expressing cells in the clusters.Furthermore,the intra-cellular insulin content was detected by RIA after the induction culture.Conclusion:These cells represent a previously unidentified adult intrinsic pancreatic precursor population and are a promising candidate for cell-based therapeutic strategies.