成年大鼠骨髓基质细胞转分化为胰岛样细胞的实验研究

目的 探讨二甲基亚枫(DMSO)对成年大鼠骨髓基质干细胞(MSC)定向诱导分化为胰岛样细胞团的作用.方法 用长骨骨髓腔冲洗的方法对Wistar大鼠MSC进行分离和培养,加入1%DMSO诱导3 d后,HDMEM培养7～10 d.通过形态学观察、双硫棕(DTZ)染色、免疫细胞化学染色、葡萄糖刺激实验和RT-PCR,对诱导细胞进行体外结构和功能鉴定.结果 1%DMSO诱导后第3 d有胰岛样结构出现;免疫细胞化学显示,诱导后第1 d MSC呈nestin免疫反应阳性,诱导后第10 d细胞团表达胰岛素、胰高血糖素,而未诱导组细胞不表达上述蛋白;RT-PCR结果表明,诱导后第10 d检测到胰岛素1(Ins1)、胰岛素2(Ins2)、胰高血糖素(glu)和生长抑素(SS)基因的表达.结论 体外经DMSO诱导,大鼠骨髓基质干细胞可定向分化为胰岛样细胞.

EXPERIMENTAL STUDY OF ISLET-LIKE CELLS TRANSDIFFERENTIATED FROM BONE MARROW STROMAL CELLS

Objective To explore the possibility of differentiating functional islet-like masses from bone marrow stromal cells induced by DMSO. Methods Adult Wistar rat bone marrow stromal cells(MSC) were isolated and cultured then induced for 3 days with serum free H-DMEM media supplemented with 1% DMSO.The cells were then cultured in H-DMEM supplemented with 10% FBS for 7-10 additional days.Morphological changes of the induced cells were observed under an inveted microscope.DTZ staining,immunocytochemical stainings were conducted to examine the production of insulin and glucagons.Glucose challenge and radio immunoassay were perfomed to evaluate the function of the aggregates,and RT-PCR was conducted to detect the expression of endocrine gene. Results While MSC cells were typically fibrocyte-like,grape-like coherent clustered cells were observed in experiment group on the 3rd day after the inducement of DMSO,which were similar to pancreatic islets.DTZ staining of the cell aggregates was positive as the natural pancreatic islet.Immunocytochemistry also confirmed that these aggregates were positive for nestin on the first day after the inducement of DMSO,and positive for insulin,glucagen on the 10th day after the inducement;while the control group were negatively stained for the characteristics of endocrine.RT-PCR showed the insulin1,insulin2,glucagon and somatostaitin gene expression on the 10th day after the inducement of DMSO.Conclusion Islet-like masses can be transdifferentiated from bone marrow cells by the inducement of DMSO.