NGFβ/HIF-1α双重转染的骨髓间充质干细胞对神经元轴突再生的作用

庄培袁; 吕刚; 范仲凯; 李永明; 张玉强; 贾志强

doi:10.3969/j.issn.2095-5227.2014.11.017

NGFβ/HIF-1α双重转染的骨髓间充质干细胞对神经元轴突再生的作用

Effect of NGFβ/HIF-1α double transfected BMSCs on neuron axonal regeneration

摘要

摘要: 目的观察神经生长因子β(nerve growth factor β，NGFβ)/低氧诱导因子-1α(hypoxia inducible factor-1α，HIF-1α)双重转染的骨髓间充质干细胞(bone marrow mesenchymal stem cells，BMSCs)对神经元轴突再生微环境的改善作用，为完善微环境中轴突再生的理论提供资料。方法分别构建携带编码NGFβ、HIF-1α的慢病毒载体，用最佳感染复数(multiplicity of infection，MOI)转染BMSCs后，Western blot检测转染后的BMSCs表达NGFβ、HIF-1α的情况。培养SD大鼠皮质神经元并将其与转染后的BMSCs用Transwell双层培养板构建共培养体系。将共培养体系在厌氧培养罐中培养48 h后，用ELISA方法检测神经元培养上清液中NGFβ、HIF-1α、血管内皮生长因子(vascular endothelial growth factor，VEGF)的表达水平，并用Image pro-Plus软件测量神经元轴突的长度。实验分组：A组：单独培养神经元；B组：BMSCs与神经元共培养；C组：NGFβ转染的BMSCs与神经元共培养；D组：HIF-1α转染的BMSCs与神经元共培养；E组：NGFβ/HIF-1α双重转染的BMSCs与神经元共培养。结果 Le-RFP-NGFβ和Le-GFP-HIF-1α基因转染BMSCs转染率分别为84.83%和89.63%。Western blot检测显示，转染后的BMSCs能表达目的蛋白NGFβ和HIF-1α。共培养体系ELISA检测结果显示，C组和E组的NGFβ的表达量明显高于A、B组P＜ 0.05)；D组和E组的HIF-1α的表达量明显高于A、B组P＜ 0.05)。D、E两组的VEGF表达量高于A、B两组P＜ 0.05)。Image pro-Plus测量神经元轴突长度结果显示，C、D、E组大于A、B两组P＜ 0.05)，E组大于C、D组P＜ 0.05)。结论与NGFβ/HIF-1α双重转染BMSCs共培养的神经元轴突生长情况良好，在共培养环境中对神经元存活发挥重要作用的NGFβ、HIF-1α、VEGF等因子高表达。

Abstract: Objective To observe the improvement of axonal regeneration microenvironment affected by NGFβ/HIF-1α double gene transfected bone marrow mesenchymal stem cells, and provide information about neuron axonal regeneration in microenvironment. Methods Lentiviral vector encoding NGFβ and HIF-1α were constructed and MOI were used to transfect BMSCs, then the expression of NGFβ and HIF-1α were detected by using western blot. Co-culture system with transwell double plates which include neurons and transfected BMSCs were constructed and then it was put in anaerobic tank for 48 hours, the expression of NGFβ, HIF-1α and VEGF in culture medium were detected by ELISA and the length of axons were measured by using Image pro-Plus software. There were five groups in this study, Group A: neuron without BMSCs; Group B: neuron with BMSCs; Group C: neuron with NGFβ transfected BMSCs; Group D: neuron with HIF-1α transfected BMSCs; Group E: neuron with NGFβ/HIF-1α double transfected BMSCs. Results The transfection efficiency of Le-RFP-NGF and Le-GFP-HIF-1α was 84.83% and 89.63%, respectively. Western blot analysis showed that transfected BMSCs could express the target protein NGFβ and HIF-1α. ELISA analysis of the co-culture system showed that the expression of NGFβ in Group C and Group E was significantly higher than in Group A and Group B (P＜ 0.05), the expression of HIF-1α in Group D and Group E was significantly higher than in Group A and Group B (P＜ 0.05). The expression of VEGF in Group D and Group E was significantly higher than in Group A and Group B. Image pro-Plus measurement showed that axon length of Group C, Group D and Group E was greater than that of Group A and Group B (P＜ 0.05), furthermore, axon length of Group E (neuron with NGFβ/HIF-1α double transfected BMSCs) was greater than that of Group C and Group D (P＜ 0.05). Conclusion The axons co-cultured with NGFβ/HIF-1α double transfected BMSC grow well and NGFβ, HIF-1α and VEGF, which play important roles in neuronal survival, are highly expressed in the coculture environment.

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