慢病毒介导的EphrinB2基因转染大鼠骨髓间充质干细胞表达的实验研究

目的将携带Ephrin B2基因慢病毒转染至骨髓间充质干细胞(BMSCs),检测其过表达水平及细胞形态学变化,并探讨Eph B4/Ephrin B2是否具有促进大鼠BMSCs体外迁移活性的作用。方法单纯贴壁法分离大鼠BMSCs,倒置显微镜观察细胞生长形态。携带Ephrin B2慢病毒以感染复数3和10感染BMSCs分为低浓度转染组(MOI=3)和高浓度转染组(MOI=10),对照组采用未转染组、阴性转染组,利用q PCR检测Ephrin B2基因表达及Western blot检测Ephrin B2蛋白水平。观察Ephrin B2基因转染后BMSCs形态学变化。15 d后通过免疫荧光检测MAP2表达了解细胞分化。进一步Transwell实验检测细胞迁移能力来了解Eph B4/Ephrin B2在调节干细胞迁移的作用。结果培养BMSCs为多角形或棱形呈漩涡状排列生长。Ephrin B2-BMSCs经q PCR和Western blot检测证实有外源性Ephrin B2表达。Ephrin B2基因转染后BMSCs 24 h,BMSCs胞体开始收缩细胞有突起伸出,72 h后可见典型的神经样细胞形态改变。15 d后,BMSCs分化成为MAP2神经元,与阴性转染组相比,低浓度转染组和高浓度转染组MAP2表达率上升(P0.05)。Transwell实验结果显示:低浓度转染组与高浓度转染组较未转染组及阴性转染组穿膜细胞数量明显增加(P0.05)。结论慢病毒介导Ephrin B2能高效感染BMSCs,并随着时间延长分化成神经样细胞,同时Eph B4/Ephrin B2在调节BMSCs迁移具有重要作用。

Experimental study of lentiviral vector-mediated EphrinB2 gene transfection rat bone marrow mesenchymal stem cells

Objective To detect the over-expression and morphological changes after EphrinB2 gene transfected to BMSCs and study the effect of EphB4/EphrinB2 on rat BMSCs in vitro.Methods The simple adherent method was adopted in isolating and culturing experiments of BMSCs.The inverted microscope was used to observe cells.Lentivirus carrying EphrinB2 infected BMSCs(MOI=10) and (MOI=3),using qPCR and Western blot to detect mRNA and protein's expression of EphrinB2 after transfection.Morphological changes of BMSCs after differentiation was observed.In 15 days,the cell differentiation was determined by MAP2 immunofluorescent staining.The migration rate was assessed to study the role of EphB4/EphrinB2 pathway in the stem cells migration by transwell chambers.Results The morphological characteristics of major of the BMSCs were unified into polygonal or fusiform,germinated into a spiral shape.We confirmed exogenous EphrinB2 expression in EphrinB2-BMSCs by qPCR and Western blot.3 days after EphrinB2 gene transfection,BMSCs cell body began to shrink,refraction enhanced cell protrusions,and differentiate into the typical neuron-like cell,qPCR and Western blot detection of nestin positive expression.In 15 days,expression levels of MAP2 in the low and high concertration transfected group were significantly higher than those in the negative control group (P<0.05).Transwell assay showed the cell number of Transmembrane in the low and high concertration transfected group were obviously higher compared to non-transfected group and negative control group (P<0.05).Conclusion Lentiviral-mediated EphrinB2 can efficiently infect BMSCs,and differentiate into neuron-like cells.EphB4/EphrinB2 pathway can play the role of migration in bone mrrow mesenchymal stem cells.