骨髓间充质干细胞培养液对大鼠皮层神经元氧糖剥夺损伤的保护作用及机制

Effect and mechanism of conditioned medium from bone marrow-derived mesenchymal stem cells on the survival of cortical neurons followed oxygen－glucose deprivation injury

摘要:

目的：探讨骨髓间充质干细胞（bone marrow-derived mesenchymal stem cells，BMSCs）培养液对大鼠皮层神经元氧糖剥夺（oxygen-glucose deprivation，OGD）损伤后细胞存活的作用及其影响机制。方法：分别取清洁级6~8周60~80 g SD大鼠和孕16~18 d孕鼠进行BMSCs和皮层神经元培养。培养第5天的皮层神经元建立OGD损伤模型，实验随机分为4组：氧糖剥夺组（OGD组）；氧糖剥夺+BMSCs培养液组（OGD+CM组），神经元OGD后培养基更换为BMSCs培养液（CM）继续培养；OGD+CM+LY294002组及OGD+LY294002组，以上2组分别加入PI3K/AKT特异性抑制剂LY294002。各组培养12 h后分别通过电子显微镜和光学显微镜观察神经元形态结构。各组培养2 h后分别检测AKT、pAKT（Ser473）、激活型Casepase-3蛋白表达量，在培养48 h后检测神经元的存活率。结果：OGD培养后的神经元突起断裂、缩短、溶解，线粒体、内质网细胞器肿胀。OGD+LY294002组、OGD+CM组分别与OGD组相比，OGD+LY294002组的细胞存活率和pAKT（Ser473）蛋白表达降低（t=3.679，P=0.021；t=2.938，P=0.042），而激活型Casepase-3蛋白表达升高（t=4.733，P=0.009）；OGD+CM组的细胞存活率和pAKT（Ser473）蛋白表达升高（t=6.630，P=0.003；t=3.288，P=0.030），而激活型Casepase-3蛋白表达降低（t=3.454，P=0.026）。与OGD+CM组相比，OGD+CM+LY294002组细胞存活率和pAKT（Ser473） 蛋白表达降低（t=14.255，P=0.000；t=3.872，P=0.018），而激活型Casepase-3蛋白表达升高（t=6.699，P=0.003）。结论：BMSCs对OGD损伤后的神经元具有保护作用，其作用机制可能与PI3K/AKT信号通道的激活相关。

Abstract:

Objective：To explore the effect and mechanism of conditioned medium from bone marrow-derived mesenchymal stem cells（BMSCs） on the survival of neurons after oxygen-glucose deprivation（OGD） injury. Methods：BMSCs and neurons were respectively cultured from 6 to 8 weeks（60 to 80 g） or pregnant 16 to 18 days Sprague-Dawley rats. After culturing for 5 days，primary cultured neurons were collected to build the model of OGD. Then experiments were randomly divided into four groups，including OGD group，OGD+CM group（the medium of neuron was replaced by culture medium of BMSCs after oxygen-glucose deprivation），OGD+CM+LY294002 group，OGD+LY294002 group（inhibitor LY294002 of PI3K/AKT signaling pathways were added to the above of two groups）. Morphology of neurons were observed by electron and light microscope after culturing for 12 h later in 4 groups. All the groups of neurons were cultured for 2 h later，and the protein levels of active Casepase-3 and pAKT（Ser473） were detected by Western bolt. After 48 h later，the survival rate of neurons was analyzed. Results：The neurite of neurons followed OGD injury were destructive and partially disappeared. Organelle of neurons，such as endoplasmic reticulum and mitochondrion，were swelled. In OGD+LY294002 group，the survival rate of neurons and expression level of pAKT（Ser473） were decreased（t=3.679，P=0.021；t=2.938，P=0.042），while the level of active Casepase-3 was increased（t=4.733，P=0.009） compared with those in OGD+CM group. The survival rate of neurons and expression level of pAKT（Ser473） were increased（t=6.630，P=0.003；t=3.288，P=0.030），while the level of active Casepase-3 was decreased（t=3.454，P=0.026） in OGD+CM group than in OGD+LY294002 group. In comparison with OGD+CM，the survival rate and level of pAKT（Ser473） were decreased（t=14.255，P=0.000；t=3.872，P=0.018） in OGD+CM+LY294002 group，while level of active Casepase-3 was increased（t=6.699，P=0.003）. Conclusion：BMSCs promote the survival of neurons after OGD damage，and its mechanisms may be related to the activation of PI3K/AKT protein signaling pathway.