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

目的观察神经生长因子β(nerve growth factorβ,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。

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.