Osteogenic and angiogenic ability of bone marrow mesenchymal stem cells synergistically over-expressing hypoxia-inducible factor-1α and bone morphogenetic protein-6 genes under hypoxia

doi:10.3969/j.issn.2095-4344.1701

Abstract

Abstract:

BACKGROUND: Hypoxia-inducible factor-1α (HIF-1α) is currently considered to be one of the most important regulators of oxygen homeostasis in the body. Bone morphogenetic protein-6 (BMP-6) is one of the most osteogenic bone morphogenetic proteins. In this study we attempted to construct bone marrow mesenchymal stem cell (BMSC) lines co-expressing HIF-1α and BMP-6 to explore the tolerance and functional benefit of the cells under hypoxic conditions.
OBJECTIVE: To investigate the proangiogenic and osteogenic ability of BMSCs synergistically over-expressing HIF-1α and BMP-6 under hypoxic environment in vitro.
METHODS: BMSCs were isolated, cultured and identified from Sprague-Dawley rats. BMSCs co-overexpressing HIF-1α and BMP-6 were constructed by adeno-associated viral vector. aav-HIF-1α-BMP-6-BMSCs were cultured under normoxia (21% O₂) and hypoxia (2% O₂), and non-transfected BMSCs under normoxia were used as a control group. mRNA levels of HIF-1α, VEGF, BMP-6, GLUT-1, SIRT-1, OCN, RUNX2, AKP were detected by RT-qPCR. After co-culture of BMSCs and human umbilical vein endothelial cells, the in vitro tube formation ability was observed. After osteogenic induction, alkaline phosphatase staining and alizarin red staining were used to identify the osteogenic effect of the cells.
RESULTS AND CONCLUSION: (1) Flow cytometry results showed that BMSCs at passage 4 highly expressed CD29, CD90 and CD44, but lowly expressed CD45, CD11 and CD34. (2) The expression levels of VEGF, GLUT-1, SIRT-1, OCN, RUNX2, and AKP mRNA in the hypoxia group were significantly higher than those in the normoxia group (P < 0.01). (3) In vitro tube formation assay results showed that the number of tubules formed in the hypoxia group was significantly higher than that in the normoxia group (P < 0.01). (4) More calcium nodules and round mineralized nodules appeared in the hypoxia group than in the normoxia group. These findings suggest that BMSCs co-expressing HIF-1α and BMP-6 can promote the osteogenic and angiogenic ability under hypoxia in vitro.

Key words: bone marrow mesenchymal stem cells, hypoxia-inducible factor-1α, bone morphogenetic protein-6, hypoxia, osteogenic ability, angiogenic ability, Natural Science Foundation of Guangdong Province

CLC Number:

Liao Hongxing, Zhang Zhihui, Liu Zhanliang, Huang Jian, Shen Yeguang, Huang Yingmei, Zhong Zhixiong. Osteogenic and angiogenic ability of bone marrow mesenchymal stem cells synergistically over-expressing hypoxia-inducible factor-1α and bone morphogenetic protein-6 genes under hypoxia[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(17): 2644-2650.

Figures/Tables 1

2.1 骨髓间充质干细胞鉴定结果流式细胞仪检测结果显示第4代骨髓间充质干细胞CD29、CD90、CD44、CD45、CD11、CD34阳性率分别为98.2%，99.1%，98.9%，1.26%，1.57%，1.91%，见图1。细胞高表达CD29、CD90、CD44，低表达CD45、CD11、CD34，表明实验所分离培养的细胞为间充质干细胞而非造血干细胞。 2.2 RT-qPCR检测转染后骨髓间充质干细胞中低氧诱导因子1α和骨形态发生蛋白6的表达 AAV-HIF-1α组低氧诱导因子1α mRNA表达水平均高于AAV-GFP组，差异有显著性意义(P < 0.05)，AAV-HIF-1α-BMP-6组HIF-1α mRNA表达水平更高，明显高于AAV-GFP组(P < 0.01)；AAV-BMP-6组骨形态发生蛋白6 mRNA表达水平均高于AAV-GFP组，差异有显著性意义(P < 0.05)，AAV-HIF-1α-BMP-6组骨形态发生蛋白6 mRNA表达水平更高，明显高于AAV-GFP组(P < 0.01)，见图2。结果表明重组AAV-HIF-1α-BMP-6转染骨髓间充质干细胞对于外源基因的表达具有协同作用。 2.3 RT-qPCR检测不同氧张力下骨髓间充质干细胞中成骨和成血管相关基因的表达常氧组细胞中VEGF、GLUT-1、SIRT-1、OCN、RUNX2、AKP mRNA的表达水平高于对照组，差异有显著性意义(P < 0.05)；低氧组细胞中VEGF、GLUT-1、SIRT-1、OCN、RUNX2、AKP mRNA表达水平更高，差异有非常显著性意义(P < 0.01)，见图3。体外低氧培养后重组AAV-HIF-1α-BMP-6-BMSCs可以促进VEGF、GLUT-1、SIRT-1、OCN、RUNX2、AKP等成骨和成血管相关基因的表达。 2.4 体外小管形成实验结果重组骨髓间充质干细胞在常氧及低氧状态下与人脐静脉上皮细胞共培养12 h后，检测小管形成能力。结果发现，常氧组的小管形成数量为(14.0±3.6)/视野，显著高于对照组(7.67±2.08)/视野，差异有显著性意义(P < 0.05)；低氧组小管形成数量为(23.0±3.0)/视野，非常显著高于对照组，差异有非常显著性意义(P < 0.01)，见图4。结果表明低氧状态下培养的重组AAV-HIF-1α-BMP-6转染骨髓间充质干细胞具有更强的体外血管生成能力。 2.5 体外成骨实验结果茜素红染色结果显示，与对照组比较，常氧组和低氧组有较多的钙结节，其中低氧组钙结节最多，细胞呈集落生长，细胞间产生致密的不透光区域，大块片状红染区形成，细胞表现明显的成骨活性。碱性磷酸酶染色结果显示，低氧组比常氧组出现更多的圆形矿化结节，表现为强阳性，见图5。茜素红染色及碱性磷酸酶染色提示低氧环境下培养的重组AAV-HIF-1α-BMP-6转染骨髓间充质干细胞表现出更强的体外成骨矿化能力。"

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