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

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

作　　者：房佰俊[1] 宋永平[1] 曹莹[1] 林全德[1] 买玲[1] 

机构地区：[1]河南省肿瘤医院河南省血液病研究所

出　　处：《西安交通大学学报（医学版）》2007年第2期138-141,共4页Journal of Xi’an Jiaotong University（Medical Sciences）

基　　金：河南省杰出青年基金(No.0612000900);河南省医学科技创新人才工程资助项目(No.200590)

摘　　要：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、碱性成纤维细胞生长因子(bFGF)及表皮生长因子(EGF)的培养基中诱导,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细胞向胰岛样细胞分化。用RT-PCR法检测诱导前后nestin、ngn3、胰岛素启动子因子1(IPF-1)、胰岛素及胰高血糖素基因的表达;免疫荧光染色法检测诱导前后nestin、胰岛素及胰高血糖素的表达;放射免疫分析法检测诱导前后细胞分泌胰岛素情况。结果成人脂肪源Flk1+CD31-CD34-细胞经第一阶段的诱导后可分化成nestin阳性的祖细胞,继续诱导6 d后变圆的细胞逐渐增多,并最终聚集成团。免疫荧光实验证明经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;放免分析结果表明,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糖尿病的细胞治疗。Objective To promote the differentiation of Flk1＋CD31^-CD34^- cells isolated from adult adipose tissues into 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 culture medium was changed. The glucose concentration in the serum-free medium was increased. At the same time, betacellulin and nicotinamide were added. Reverse transcription polymerase chain reaction （RT-PCR） was used to detect the expression of nestin, ngn3, insulin promoter factor-1 （IPF-1）, insulin, and glucagon before and after differentiation induction; immunofluorescent staining for nestin, insulin and glucagon and radioimmunoassay （RIA） for insulin. Results Initially, a nestin positive precursor cell population was found, then small round cells increased in number after 6 days. Later on, they were differentiated into islet-like clusters. The induced 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 intracellular 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.

关 键 词：干细胞 胰岛 脂肪 

分 类 号：Q254[生物学—细胞生物学] R322.57[医药卫生—人体解剖和组织胚胎学]