Abstract:

Objective To investigate the level of hypoxia inducible factor-1α (HIF-1α) on osteoblasts and angiogenesis-associated cytokines in bone marrow mesenchymal stem cells (BMSCs) from SD rats.Methods BMSCs were isolated and cultured and identified by flow cytometry. Plasmid vectors containing upregulated and downregulated HIF-1α gene and a control vector were constructed. The plasmids were transfected into BMSCs by Lipofectamine^?LTX transfection reagent, and the cells were divided into an overexpression experimental group, an overexpression control group, a low expression experimental group and a low expression control group. All components were stained with a lizarin red 3 d and 7 d after osteogenesis induction. The mRNA expression levels of the target gene HIF-1α, osteogenic differentiation-specific markers, including Runt-related transcription factor 2 (Runx2) and angiogenic markers, including platelet-derived growth factor-BB (PDGF-BB) and transforming growth factor-β (TGF-β), were detected by RT-PCR. Western blot was used to detect the protein expression of the target proteins HIF-1α, Runx2, and PDGF-BB.Results The CD29- and CD45-positivity rates of BMSC surface markers identified by flow cytometry were 98.2% and 4.2%, respectively. RT-PCR results showed that the mRNA expression of HIF-1α, Runx2, TGF-β and PDGF-BB was observably increased (P < 0.001). The mRNA expression levels of HIF-1α, Runx2, TGF-β and PDGF-BB in BMSCs from the low expression experimental group were significantly reduced (P < 0.001). Western blot results showed that the expression levels of HIF-1α, Runx2 and PDGF-BB in BMSCs from the overexpression experimental group were all increased (P < 0.001). The expression levels of HIF-1α, Runx2 and PDGF-BB in BMSCs from the low expression experimental group were reduced (P < 0.001). Alizarin red staining results showed that the area of calcium nodules in the low expression experimental group was smaller than that in low expression control group, the area of red calcium nodules in the over expression experimental group was larger than that in over expression control group, and with the increase of osteogenic induction time, the calcification area of each group also increased.Conclusion Upregulation and downregulation of HIF-1α can regulate the osteogenic differentiation and the expression of angiogenesis related factors of BMSCs.

Key words: hypoxia inducible factor-1α, bone marrow mesenchymal stem cells, osteogenic differentiation, angiogenesis, Runt-related transcription factor 2, platelet derived growth factor-BB, transforming growth factor-β, phosphatidylinositol 3-kinase, protein kinase B, bone tissue engineering

摘要：

目的 探讨低氧诱导因子-1α（hypoxia-inducible factor-1α,HIF-1α）在SD大鼠骨髓间充质干细胞（bone marrow mesenchymal stem cells,BMSCs）中对成骨分化和血管生成相关细胞因子的影响。方法 分离并培养BMSCs,采用流式细胞术进行鉴定;分别构建上调和下调HIF-1α基因的质粒载体及对照组质粒,使用Lipofectamine^?LTX转染试剂将各组质粒分别转染至BMSCs,将细胞分为过表达实验组、过表达对照组、低表达实验组和低表达对照组。各组成骨诱导3、7 d后分别进行茜素红染色;RT-PCR检测目的基因HIF-1α,成骨分化特异标志物Runt相关转录因子2（Runt-related transcription factor 2,Runx2）,以及血管生成特异标志物血小板衍生生长因子-BB（platelet derived growth factor-BB,PDGF-BB）、转化生长因子-β（transforming growth factor-β,TGF-β）的mRNA表达水平;用Western blot检测目的蛋白HIF-1α及Runx2、PDGF-BB的蛋白表达量。结果 流式鉴定BMSCs表面标记物CD29、CD45阳性表达率分别为98.2%和4.2%;RT-PCR结果显示：过表达实验组BMSCs中HIF-1α、Runx2、TGF-β和PDGF-BB的mRNA表达显著升高（P < 0.001）;低表达组中HIF-1α、Runx2、TGF-β 和PDGF-BB的mRNA表达显著降低（P < 0.001）;Western blot结果显示：过表达实验组BMSCs中HIF-1α、Runx2和PDGF-BB的蛋白表达量均增高（P < 0.001）,低表达实验组中HIF-1α、Runx2和PDGF-BB蛋白表达量降低（P < 0.001）。茜素红染色结果显示,低表达实验组钙结节面积小于其对照组,过表达实验组红色钙结节面积显著大于其对照组,随着成骨诱导时间的增加,各组钙化面积也增大。结论 上调和下调HIF-1α后可调控BMSCs的成骨分化及血管生成相关因子表达。

关键词: 低氧诱导因子-1α, 骨髓间充质干细胞, 成骨分化, 血管生成, Runt相关转录因子2, 血小板衍生生长因子-BB, 转化生长因子-β, 磷脂酰肌醇3-激酶, 蛋白激酶B, 骨组织工程