金雀异黄素对骨髓间充质干细胞诱导分化为神经元样细胞前后蛋白和Ca^(2+)浓度变化的研究  

The study on the effects of genistein on the changes of protein expression and calcium ion concentration of mesenchymal stem cells before and after differentiating into neuron-like cells

作　　者：潘国强 齐凤平 王相利 

机构地区：[1]河北省迁安市人民医院,064400

出　　处：《河北医药》2013年第1期7-9,共3页Hebei Medical Journal

摘　　要：目的探讨不同浓度金雀异黄素对骨髓间充质干细胞(Mesenchymal Stem Cells,MSCs)按照Woodbury经典诱导方案将其诱导分化为神经元样细胞前后蛋白和细胞内游离Ca2+浓度的变化。方法分离纯化SD大鼠MSCs进行传代培养,传至第5代时,待细胞融汇成致密单层后,加入不同浓度金雀异黄素(0、25、50、75和100μmol/L)培养24h后,按照Woodbury经典诱导方案进行诱导,采用免疫组化技术检测诱导前后GAP-43、NSE、NF和Nestin的蛋白表达的变化,采用激光扫描共聚焦显微镜检测诱导前后细胞内游离钙离子浓度的变化。结果随着金雀异黄素浓度的增加,诱导后各组GAP-43、NSE、NF和Nestin蛋白表达水平逐渐增高。更换诱导液后,各组细胞内荧光强度逐渐增加,到100s达高峰值,其后逐渐减弱,但20min时细胞荧光强度仍高于诱导前,但是随着金雀异黄素浓度的增加,细胞内游离Ca2+浓度呈现浓度依赖性下降。结论金雀异黄素能够促进MSCs诱导分化为神经元样细胞。游离Ca2+在诱导过程中可能不起关键性作用。Objective To investigate the effects of different doses of genistein on the changes of protein expression and calcium ion concentration of marrow mesenehymal stem ceils （MSCs） before and after the cells were induced into neuron-like cells by means of Woodbury classic induction schema. Methods The MSCs of SD rats were separated and purified, then the cells were subcultured till the fifth generation, after MSCs grew and became into compacted monolayer, the MSCs were treated with 0,25,50,75 and 100 μmol/L genistein for 24 h, respectively, next the MSCs were induced into neuron-like cells by means of Woodbury classic induction schema. The protein expression levels of GAP-43, NSE, NF and Nestin were detected by immunocytochemistry, and the changes of intracellular free Ca2 ＋ concentration before and after induction were detected by laser scan confocal microscope. Results After MSCs were induced, the expression levels of GAP-43, NSE, NF and Nestin were increased with the increase of genistein concentration （ P 〈 0.05 ）. The fluorescence intensity in MSCs was increased gradually in every group after the culture medium was replaced with the induction medium ,which reached peak at 100s, then decreased gradually, however, at 20min which was still higher than that before the induction. The intracellular free Ca2＋ concentration was increased by genistein in a dose-dependent manner （ P 〈 0. 05 ）. Conclusion Genistein can promote and induce MSCs to differentiate into neuron-like cells. The intraeellular free Ca2＋ may not play an important role in the course of induction.

关 键 词：骨髓间充质干细胞 神经元样细胞 免疫组化 激光扫描共聚焦显微镜 细胞内游离钙离子 

分 类 号：R551.3[医药卫生—血液循环系统疾病]