大鼠骨髓间质干细胞体外诱导分化为胰岛素分泌细胞的实验研究

IN VITRO TRANSDIFFERENTIATION OF RAT BONE MARROW MESENCHYMAL STEM CELLS INTO INSULIN-SECRETING CELLS

目的研究体外定向诱导大鼠骨髓间质干细胞(BMSCs)横向分化为胰岛素分泌细胞的可能性,并了解该细胞是否具备分泌胰岛素和胰高血糖素的功能。方法从健康成年大鼠骨髓中分离BMSCs,通过传代对细胞进行纯化和扩增。采用两期诱导方案,用诱导液1使BMSCs向巢蛋白(nestin)阳性的祖细胞分化;用诱导液2使nestin阳性的祖细胞向胰岛素分泌细胞分化。用双硫腙染色鉴定胰岛素分泌细胞团;免疫细胞化学法检测诱导前后nestin、胰岛素及胰高血糖素蛋白的表达;采用放射免疫分析法检测葡萄糖刺激后上清中胰岛素的量。结果BMSCs经第一期诱导5d可分化成nestin阳性的祖细胞,双硫腙染色阳性。免疫细胞化学显示,经两期,诱导后的细胞表达胰岛素、胰高血糖素等激素。放射免疫分析结果显示,诱导后的细胞团可以分泌胰岛素,糖反应性较弱。结论健康成年大鼠骨髓间质干细胞在体外可以被诱导分化为胰岛样细胞团,且具有可重复性。

Objective To investigate the possibility of bone marrow mesenchymal stem cells to transdifferentiate into insulinf-secreting cells or islet progenitors, and to demonstrate whether these islet-like cells can generate insulin and glucagon. Methods BMSCs were isolated from tibia or thighbone of 6-8 weeks normal SD rat, purified on the basis of their ability to adhere to matrix, and expanded through their self-renewal. Two-step strategy was adopted, BMSCs were induced to generate nestin-positive cells under diffferentiation culture medium 1（added 20μg/L bFGF, 10 μg/L EGF and 2% B27 in DMDM/F12 medium） in step one; Nestin-positive cells were differentiated and developed into islet-like cells or islet progenitors under complexed culture medium 2（added 10 μg/L Betacellulin, 10 μg/L HCF, 10 μg/L Activin A, 10 mmol/L nicotide and 2% B27 serum-free in HG-DMEM medium） in step two. The differentiated cells were tested by Dithiazone staining; Immunocytochemistry was carried out using standard protocols in which the cells were incubated with Rb-Rat insulin monoclone antibody, mouse-human glucogan polyclone antibody and Rb-Rat nestin nronoclone antibody and assessed by examining the cells under a Zeiss microscope. Insulin secretion in response to insulin secretagogues （glucose） was assayed by radioimmunoassay （RIA）. Results After 5 days, BMSCs were expressing nestin, a marker shared with neural stem cells and pancreatic stem cells. Dithiazone staining was positive （bright orange）. The differentiated cell masses were containing insulin and glycagon as indicated by immunocytochemistry. RIA demonstrated that these cell mass could generate insulin, the responsibility to glucose was weak. Conclusion BMSCs can be induced to differentiate into insulin-secreting cells or islet progenitors by adding various different cell growth factors and the strategy is repeatable. This study may present a potential approach for the treatment of insulin-depended diabetes by interventional therapy.