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

发布时间：2018-04-09 20:04

本文选题：EphrinB　切入点：EphB　出处：《中风与神经疾病杂志》2017年06期

【摘要】：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。
[Abstract]:Objective to transfect lentivirus carrying Ephrin B2 gene into bone marrow mesenchymal stem cells (BMSCs) to detect its overexpression level and cell morphology, and to investigate whether Eph B4/Ephrin B2 can promote the migration of rat BMSCs in vitro.Methods BMSCs were isolated from rats by simple adherent method, and cell growth morphology was observed by inverted microscope.Ephrin B2 lentivirus was divided into low concentration transfection group and high concentration transfection group by infecting BMSCs with complex number 3 and 10. The control group was used to detect the expression of Ephrin B2 gene by Q PCR and the Western blot to detect Ephrin B2 protein level by using untransfected group and negative transfection group.The morphological changes of BMSCs were observed after Ephrin B2 gene transfection. After 15 days, the expression of MAP2 was detected by immunofluorescence to understand the cell differentiation.Further Transwell assay was used to investigate the role of Eph B4/Ephrin B2 in regulating stem cell migration.Results the cultured BMSCs were polygonal or prism in swirl shape. Ephrin B2-BMSCs was confirmed by Q PCR and Western blot to express Ephrin B2. Ephrin B2 gene was transfected into the cell body of BMSCs 24 h after transfection, the cell body began to contract. 72 h later, typical cells could be seen.After 15 days, BMSCs differentiated into MAP2 neurons.Compared with the negative transfection group, the expression rate of MAP2 in the low concentration transfection group and the high concentration transfection group was higher than that in the low concentration transfection group and the high concentration transfection group. The results showed that the number of the perforating cells in the low concentration transfection group and the high concentration transfection group was significantly higher than that in the non-transfection group and the negative transfection group.Conclusion Lentivirus-mediated Ephrin B2 can efficiently infect BMSCs and differentiate into neuron-like cells over time. Meanwhile, Eph B4/Ephrin B2 plays an important role in regulating the migration of BMSCs.
【作者单位】：南京医科大学附属儿童医院康复科;南京大学医学院附属鼓楼医院神经内科;
【基金】：国家自然科学基金青年基金(No.81401864) 南京医科大学科学基金重点项目(No.2013NJMU097)
【分类号】：R329.2

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