缺氧预处理激活HIF-1α/MALAT1/VEGFA通路促进骨髓间充质干细胞生存和血管再生

Hypoxia preconditioning promotes bone marrow mesenchymal stem cells survival and vascularization through the activation of HIF-1α/MALAT1/VEGFA pathway

背景:既往研究发现缺氧预处理能够提高骨髓间充质干细胞生存和改善其移植治疗效率,且此效应主要由缺氧诱导因子1α(hypoxia induced factor-1α,HIF-1α)介导,但下游机制未明。目的:体外观察缺氧预处理对骨髓间充质干细胞生存和血管再生能力的影响,探讨HIF-1α/MALAT1/VEGFA通路在其中的调控作用。方法:将体外培养的骨髓间充质干细胞分为缺氧实验组(体积分数为1%O2)和常氧对照组(体积分数为20%O2),培养24 h后检测细胞增殖、凋亡以及血管形成情况以及HIF-1α、MALAT1和VEGFA的表达;在此基础上,分别采用相应的siRNA抑制HIF-1α以及MALAT1的表达,以HIF-1αsiRNA scramble和MALAT1 siRNA scramble作为阴性对照,再进行缺氧预处理,检测并比较各组通路因子的表达变化。结果与结论:①与常氧对照组相比,缺氧实验组细胞生存力明显增强,凋亡比例显著减少,血管管腔样结构明显增多(P<0.01),HIF-1α、MALAT1和VEGFA的表达均明显增高(P<0.01);②在抑制HIF-1α的基础上进行缺氧预处理,MALAT1和VEGFA表达均明显下降(P<0.01);而抑制MALAT1后,VEGFA的表达亦显著降低(P<0.01);③结果表明,缺氧预处理能够通过激活HIF-1α/MALAT1/VEGFA通路促进骨髓间充质干细胞生存和血管再生。

BACKGROUND:Previous study demonstrated that hypoxia preconditioning promoted mesenchymal stem cells survival and their therapeutic efficacy,and this effect was mediated by hypoxia induced factor-1α(HIF-1α).However,specific downstream mechanism remained unclear.OBJECTIVE:To observe the influence of hypoxia preconditioning on the survival and vascularization potential of bone marrow mesenchymal stem cells in vitro and explore the regulatory mechanism of HIF-1α/MALAT1/VEGFA pathway.METHODS:Bone marrow mesenchymal stem cells were obtained and cultured in vitro.Cells were divided into hypoxia(1%O2)and normoxia control groups(20%O2),and cultured for 24 hours.Cells proliferation,apoptosis and vascularization were evaluated.The expression of HIF-1α,MALAT1,and VEGFA was detected.HIF-1αand MALAT1 were inhibited by their siRNAs separately.HIF-1αsiRNA scramble and MALAT1 siRNA scramble were used as negative controls before hypoxia preconditioning.Alterations of the molecules were examined and compared in different groups.RESULTS AND CONCLUSION:(1)Compared with the normoxia control group,cell viability was significantly enhanced;and cell apoptosis percentage was significantly declined in the hypoxia group;vascular lumen like structure was also increased significantly in the hypoxia group(P<0.01);expression of HIF-1α,MALAT1,and VEGFA was significantly increased in the hypoxia group(P<0.01).(2)After the inhibition of HIF-1αand hypoxia preconditioning,both MALAT1 and VEGFA expression levels were significantly reduced(P<0.01).The expression of VEGFA was also significantly suppressed after the blockage of MALAT1(P<0.01).(3)This study suggested that hypoxia preconditioning effectively promoted bone marrow mesenchymal stem cell survival and vascularization through the activation of HIF-1α/MALAT1/VEGFA pathway.