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

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

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6d,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6d后可分化成nestin+的祖细胞;继续用诱导液2诱导6d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。

Aim ： To promote differentiation of Flk1^＋CD31^-CD34^- cells isolated from human adult adipose tissue toward pancreatic islet endocrine cells in vitro. Mothods：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 Next, the glucose concentration in the medium was increased, bFGF was withdrawn, betacellulin and nicotinamide was added.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.