低氧诱导因子1α与骨形态发生蛋白6协同过表达骨髓间充质干细胞在低氧环境下的成骨和成血管效应
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摘要
背景:目前认为低氧诱导因子1α是维持机体细胞内氧稳态的最重要调控因子之一,骨形态发生蛋白6是成骨活性最强的骨形态发生蛋白之一,实验拟构建二者共表达的骨髓间充质干细胞系,以研究细胞在低氧状态下的耐受及功能受益问题。目的:探讨体外模拟低氧环境下低氧诱导因子1α和骨形态发生蛋白6协同过表达骨髓间充质干细胞的成骨和成血管生物学特性。方法:分离培养鉴定SD大鼠骨髓间充质干细胞,将已构建好的携带有骨形态发生蛋白6和低氧诱导因子1α双基因的腺病毒真核表达载体共同感染骨髓间充质干细胞,在体积分数为21%O_2,2%O_2条件下分别进行培养,并以常氧状态下未转染的骨髓间充质干细胞作为对照组,RT-qPCR检测各组细胞HIF-1α、VEGF、BMP-6、GLUT-1、SIRT-1、OCN、RUNX2、AKP的m RNA水平。各组骨髓间充质干细胞与人脐静脉内皮细胞共培养后,观察体外小管形成能力;各组骨髓间充质干细胞经成骨诱导培养基干预后,碱性磷酸酶染色及茜素红染色鉴定成骨效果。结果与结论:(1)流式细胞仪检测结果显示第4代骨髓间充质干细胞高表达CD29、CD90、CD44,低表达CD45、CD11、CD34;(2)低氧组骨髓间充质干细胞VEGF、GLUT-1、SIRT-1、OCN、RUNX2、AKP m RNA表达水平高于常氧组(P<0.01);(3)低氧组小管形成数量要明显高于对照组,差异有非常显著性意义(P<0.01);(4)低氧组细胞出现更多的钙结节及圆形矿化结节;(5)上述结果表明,低氧诱导因子1α与骨形态发生蛋白6协同过表达骨髓间充质干细胞在体外低氧环境下具有更强的血管生成及成骨分化能力。
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% O_2) and hypoxia(2% O_2), 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-1αand BMP-6 can promote the osteogenic and angiogenic ability under hypoxia in vitro.
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% O_2) and hypoxia(2% O_2), 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-1αand BMP-6 can promote the osteogenic and angiogenic ability under hypoxia in vitro.
引文
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[10]Büning H,Perabo L,Coutelle O,et al.Recent developments in adeno-associated virus vector technology.J Gene Med.2008;10(7):717-733.
[11]Luo G,Huang Y,Gu F.rhBMP2-loaded calcium phosphate cements combined with allogenic bone marrow mesenchymal stem cells for bone formation.Biomed Pharmacother.2017;92:536-543.
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[14]Sharma S,Sapkota D,Xue Y,et al.Delivery of VEGFA in bone marrow stromal cells seeded in copolymer scaffold enhances angiogenesis,but is inadequate for osteogenesis as compared with the dual delivery of VEGFA and BMP2 in a subcutaneous mouse model.Stem Cell Res Ther.2018;9(1):23.
[15]Ramasamy SK,Kusumbe AP,Wang L,et al.Endothelial Notch activity promotes angiogenesis and osteogenesis in bone.Nature.2014;507(7492):376-380.
[16]Gómez-Puerto MC,Verhagen LP,Braat AK,et al.Activation of autophagy by FOXO3 regulates redox homeostasis during osteogenic differentiation.Autophagy.2016;12(10):1804-1816.
[17]Liu Y,Yang X,Maureira P,et al.Permanently Hypoxic Cell Culture Yields Rat Bone Marrow Mesenchymal Cells with Higher Therapeutic Potential in the Treatment of Chronic Myocardial Infarction.Cell Physiol Biochem.2017;44(3):1064-1077.
[18]Ciapetti G,Granchi D,Fotia C,et al.Effects of hypoxia on osteogenic differentiation of mesenchymal stromal cells used as a cell therapy for avascular necrosis of the femoral head.Cytotherapy.2016;18(9):1087-1099.
[19]Maes C,Carmeliet G,Schipani E.Hypoxia-driven pathways in bone development,regeneration and disease.Nat Rev Rheumatol.2012;8(6):358-366.
[20]Wu WJ,Zhang XK,Zheng XF,et al.SHH-dependent knockout of HIF-1 alpha accelerates the degenerative process in mouse intervertebral disc.Int J Immunopathol Pharmacol.2013;26(3):601-609.
[21]Maxwell P,Salnikow K.HIF-1:an oxygen and metal responsive transcription factor.Cancer Biol Ther.2004;3(1):29-35.
[22]李广周,吴伟.低氧环境对骨代谢影响的研究与进展[J].中国组织工程研究,2016,20(33):4963-4969.
[23]Fan L,Li J,Yu Z,et al.The hypoxia-inducible factor pathway,prolyl hydroxylase domain protein inhibitors,and their roles in bone repair and regeneration.Biomed Res Int.2014;2014:239356.
[24]Ma C,Wei Q,Cao B,et al.A multifunctional bioactive material that stimulates osteogenesis and promotes the vascularization bone marrow stem cells and their resistance to bacterial infection.PLoS One.2017;12(3):e0172499.
[25]Park HS,Kim JH,Sun BK,et al.Hypoxia induces glucose uptake and metabolism of adipose?derived stem cells.Mol Med Rep.2016;14(5):4706-4714.
[26]Yuan HF,Zhai C,Yan XL,et al.SIRT1 is required for long-term growth of human mesenchymal stem cells.J Mol Med(Berl).2012;90(4):389-400.
[27]Joo HY,Yun M,Jeong J,et al.SIRT1 deacetylates and stabilizes hypoxia-inducible factor-1α(HIF-1α)via direct interactions during hypoxia.Biochem Biophys Res Commun.2015;462(4):294-300.
[28]Xu Y,Wang S,Tang C,et al.Upregulation of long non-coding RNA HIF 1α-anti-sense 1 induced by transforming growth factor-β-mediated targeting of sirtuin 1 promotes osteoblastic differentiation of human bone marrow stromal cells.Mol Med Rep.2015;12(5):7233-7238.
[29]Chen MC,Hsu WL,Hwang PA,et al.Low Molecular Weight Fucoidan Inhibits Tumor Angiogenesis through Downregulation of HIF-1/VEGF Signaling under Hypoxia.Mar Drugs.2015;13(7):4436-4451.
[30]Uccella S,La Rosa S,Scaldaferri D,et al.New insights into hypoxia-related mechanisms involved in different microvascular patterns of bronchopulmonary carcinoids and poorly differentiated neuroendocrine carcinomas.Role of ribonuclease T2(RNASET2)and HIF-1α.Hum Pathol.2018;79:66-76.
[31]Jain T,Nikolopoulou EA,Xu Q,et al.Hypoxia inducible factor as a therapeutic target for atherosclerosis.Pharmacol Ther.2018;183:22-33.
[2]Bornes TD,Adesida AB,Jomha NM.Articular Cartilage Repair with Mesenchymal Stem Cells After Chondrogenic Priming:A Pilot Study.Tissue Eng Part A.2018;24(9-10):761-774.
[3]Wang W,Wang Y,Deng G,et al.Transplantation of Hypoxic-Preconditioned Bone Mesenchymal Stem Cells Retards Intervertebral Disc Degeneration via Enhancing Implanted Cell Survival and Migration in Rats.Stem Cells Int.2018;2018:7564159.
[4]B?hrnsen F,Schliephake H.Supportive angiogenic and osteogenic differentiation of mesenchymal stromal cells and endothelial cells in monolayer and co-cultures.Int J Oral Sci.2016;8(4):223-230.
[5]Liao H,Zhong Z,Liu Z,et al.Bone mesenchymal stem cells co-expressing VEGF and BMP-6 genes to combat avascular necrosis of the femoral head.Exp Ther Med.2018;15(1):954-962.
[6]Muinos-López E,Ripalda-Cemboráin P,López-Martínez T,et al.Hypoxia and Reactive Oxygen Species Homeostasis in Mesenchymal Progenitor Cells Define a Molecular Mechanism for Fracture Nonunion.Stem Cells.2016;34(9):2342-2353.
[7]Cox TR,Erler JT,Rumney RMH.Established Models and New Paradigms for Hypoxia-Driven Cancer-Associated Bone Disease.Calcif Tissue Int.2018;102(2):163-173.
[8]Stegen S,Deprez S,Eelen G,et al.Adequate hypoxia inducible factor 1αsignaling is indispensable for bone regeneration.Bone.2016;87:176-186.
[9]Semenza GL.Hydroxylation of HIF-1:oxygen sensing at the molecular level.Physiology(Bethesda).2004;19:176-182.
[10]Büning H,Perabo L,Coutelle O,et al.Recent developments in adeno-associated virus vector technology.J Gene Med.2008;10(7):717-733.
[11]Luo G,Huang Y,Gu F.rhBMP2-loaded calcium phosphate cements combined with allogenic bone marrow mesenchymal stem cells for bone formation.Biomed Pharmacother.2017;92:536-543.
[12]Westhauser F,H?llig M,Reible B,et al.Bone formation of human mesenchymal stem cells harvested from reaming debris is stimulated by low-dose bone morphogenetic protein-7 application in vivo.J Orthop.2016;13(4):404-408.
[13]Roth S,Dreixler JC,Mathew B,et al.Hypoxic-Preconditioned Bone Marrow Stem Cell Medium Significantly Improves Outcome After Retinal Ischemia in Rats.Invest Ophthalmol Vis Sci.2016;57(7):3522-3532.
[14]Sharma S,Sapkota D,Xue Y,et al.Delivery of VEGFA in bone marrow stromal cells seeded in copolymer scaffold enhances angiogenesis,but is inadequate for osteogenesis as compared with the dual delivery of VEGFA and BMP2 in a subcutaneous mouse model.Stem Cell Res Ther.2018;9(1):23.
[15]Ramasamy SK,Kusumbe AP,Wang L,et al.Endothelial Notch activity promotes angiogenesis and osteogenesis in bone.Nature.2014;507(7492):376-380.
[16]Gómez-Puerto MC,Verhagen LP,Braat AK,et al.Activation of autophagy by FOXO3 regulates redox homeostasis during osteogenic differentiation.Autophagy.2016;12(10):1804-1816.
[17]Liu Y,Yang X,Maureira P,et al.Permanently Hypoxic Cell Culture Yields Rat Bone Marrow Mesenchymal Cells with Higher Therapeutic Potential in the Treatment of Chronic Myocardial Infarction.Cell Physiol Biochem.2017;44(3):1064-1077.
[18]Ciapetti G,Granchi D,Fotia C,et al.Effects of hypoxia on osteogenic differentiation of mesenchymal stromal cells used as a cell therapy for avascular necrosis of the femoral head.Cytotherapy.2016;18(9):1087-1099.
[19]Maes C,Carmeliet G,Schipani E.Hypoxia-driven pathways in bone development,regeneration and disease.Nat Rev Rheumatol.2012;8(6):358-366.
[20]Wu WJ,Zhang XK,Zheng XF,et al.SHH-dependent knockout of HIF-1 alpha accelerates the degenerative process in mouse intervertebral disc.Int J Immunopathol Pharmacol.2013;26(3):601-609.
[21]Maxwell P,Salnikow K.HIF-1:an oxygen and metal responsive transcription factor.Cancer Biol Ther.2004;3(1):29-35.
[22]李广周,吴伟.低氧环境对骨代谢影响的研究与进展[J].中国组织工程研究,2016,20(33):4963-4969.
[23]Fan L,Li J,Yu Z,et al.The hypoxia-inducible factor pathway,prolyl hydroxylase domain protein inhibitors,and their roles in bone repair and regeneration.Biomed Res Int.2014;2014:239356.
[24]Ma C,Wei Q,Cao B,et al.A multifunctional bioactive material that stimulates osteogenesis and promotes the vascularization bone marrow stem cells and their resistance to bacterial infection.PLoS One.2017;12(3):e0172499.
[25]Park HS,Kim JH,Sun BK,et al.Hypoxia induces glucose uptake and metabolism of adipose?derived stem cells.Mol Med Rep.2016;14(5):4706-4714.
[26]Yuan HF,Zhai C,Yan XL,et al.SIRT1 is required for long-term growth of human mesenchymal stem cells.J Mol Med(Berl).2012;90(4):389-400.
[27]Joo HY,Yun M,Jeong J,et al.SIRT1 deacetylates and stabilizes hypoxia-inducible factor-1α(HIF-1α)via direct interactions during hypoxia.Biochem Biophys Res Commun.2015;462(4):294-300.
[28]Xu Y,Wang S,Tang C,et al.Upregulation of long non-coding RNA HIF 1α-anti-sense 1 induced by transforming growth factor-β-mediated targeting of sirtuin 1 promotes osteoblastic differentiation of human bone marrow stromal cells.Mol Med Rep.2015;12(5):7233-7238.
[29]Chen MC,Hsu WL,Hwang PA,et al.Low Molecular Weight Fucoidan Inhibits Tumor Angiogenesis through Downregulation of HIF-1/VEGF Signaling under Hypoxia.Mar Drugs.2015;13(7):4436-4451.
[30]Uccella S,La Rosa S,Scaldaferri D,et al.New insights into hypoxia-related mechanisms involved in different microvascular patterns of bronchopulmonary carcinoids and poorly differentiated neuroendocrine carcinomas.Role of ribonuclease T2(RNASET2)and HIF-1α.Hum Pathol.2018;79:66-76.
[31]Jain T,Nikolopoulou EA,Xu Q,et al.Hypoxia inducible factor as a therapeutic target for atherosclerosis.Pharmacol Ther.2018;183:22-33.