骨形态发生蛋白2和转化生长因子β2协同促进骨髓间充质干细胞成骨分化
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摘要
背景:从细胞及基因水平揭示骨形态发生蛋白2和转化生长因子β2具有协同促进骨髓间充质干细胞成骨分化的机制,为骨细胞的再生提供了实验和理论基础。目的:探讨过表达骨形态发生蛋白2和转化生长因子β2协同促进骨髓间充质干细胞成骨分化的作用。方法:通过酶消化法原代培养获取大鼠骨髓间充质干细胞,慢病毒包装骨形态发生蛋白2及转化生长因子β2的重组质粒。将携带目的基因重组质粒的慢病毒转染至第3代骨髓间充质干细胞,MTS实验检测骨髓间充质干细胞的增殖情况,定量PCR检测骨形态发生蛋白2和转化生长因子β2的m RNA表达水平,细胞爬片免疫组化检测二维混合培养条件下成骨分化标志物RUNX2在蛋白水平的表达量,体外三维培养获得的细胞团块经石蜡切片做Vonkossa染色(银染)定量分析成骨分化的阳性区域及表达强度。结果与结论:(1)转染骨形态发生蛋白2及转化生长因子β2的骨髓间充质干细胞的增殖能力与未转染的骨髓间充质干细胞相比差异无显著性意义(P> 0.05);(2)转染后骨髓间充质干细胞的骨形态发生蛋白2及转化生长因子β2 mRNA水平高于未转染的骨髓间充质干细胞,差异有显著性意义(P <0.05);(3)体外二维培养条件下细胞爬片RUNX2免疫组化及体外三维培养石蜡切片Vonkossa染色均发现共转染组骨髓间充质干细胞的成骨分化能力强于单独转染组(P <0.05);(4)结果表明,慢病毒携带骨形态发生蛋白2和转化生长因子β2的重组质粒过表达具有协同促进大鼠骨髓间充质干细胞成骨分化的作用。
BACKGROUND: It is revealed from the cell and gene level that bone morphogenetic protein 2(BMP2) and transforming growth factor β2(TGFβ2) synergistically promote the osteogenic differentiation of bone marrow mesenchymal stem cells, providing an experimental and theoretical basis for the bone regeneration. OBJECTIVE: To investigate the overexpression of BMP2 and TGFβ2 in vitro to synergistically promote the osteogenic differentiation of bone marrow mesenchymal stem cells. METHODS: Primary rat bone marrow mesenchymal stem cells were obtained by enzymatic digestion. Recombinant plasmids of BMP2 and TGFβ2 were packaged by lentivirus. The lentivirus carrying the recombinant plasmids of the target genes BMP2 and TGFβ2 was transfected into the third generation of bone marrow mesenchymal stem cells. MTS(cell proliferation assay) was used to detect the proliferation of bone marrow mesenchymal stem cells in different treatment groups at different time points. Quantitative PCR was used to detect the overexpression of BMP2 and TGFβ2 at mRNA levels. Immunohistochemistry of cell slides was performed to detect the expression level of osteogenic differentiation marker RUNX2 at the protein level under two-dimensional mixed culture conditions. The cell pellet obtained by three-dimensional culture in vitro was subjected to paraffin sectioning for Vonkossa staining(silver staining) to determine the positive region and expression intensity of osteogenic differentiation, and quantitative analysis was performed. RESULTS AND CONCLUSION:(1) The proliferation of bone marrow mesenchymal stem cells transfected with BMP2 and TGFβ2 was not statistically different from that of untransfected cells(P > 0.05).(2) The BMP2 and TGFβ2 mRNA levels were significantly higher in the transfected cells than untransfected ones(P < 0.05).(3) RUNX2 immunohistochemistry under the in vitro two-dimensional culture conditions and Vonkossa staining under the in vitro three-dimensional culture showed that the BMP2 and TGFβ2 co-transfected bone marrow mesenchymal stem cells had stronger osteogenic ability than BMP2 or TGFβ2 alone transfected cells(P < 0.05). In conclusion, the overexpression of recombinant plasmids of BMP2 and TGFβ2 carried by lentivirus synergistically promotes the osteogenic differentiation of rat bone marrow mesenchymal stem cells.
BACKGROUND: It is revealed from the cell and gene level that bone morphogenetic protein 2(BMP2) and transforming growth factor β2(TGFβ2) synergistically promote the osteogenic differentiation of bone marrow mesenchymal stem cells, providing an experimental and theoretical basis for the bone regeneration. OBJECTIVE: To investigate the overexpression of BMP2 and TGFβ2 in vitro to synergistically promote the osteogenic differentiation of bone marrow mesenchymal stem cells. METHODS: Primary rat bone marrow mesenchymal stem cells were obtained by enzymatic digestion. Recombinant plasmids of BMP2 and TGFβ2 were packaged by lentivirus. The lentivirus carrying the recombinant plasmids of the target genes BMP2 and TGFβ2 was transfected into the third generation of bone marrow mesenchymal stem cells. MTS(cell proliferation assay) was used to detect the proliferation of bone marrow mesenchymal stem cells in different treatment groups at different time points. Quantitative PCR was used to detect the overexpression of BMP2 and TGFβ2 at mRNA levels. Immunohistochemistry of cell slides was performed to detect the expression level of osteogenic differentiation marker RUNX2 at the protein level under two-dimensional mixed culture conditions. The cell pellet obtained by three-dimensional culture in vitro was subjected to paraffin sectioning for Vonkossa staining(silver staining) to determine the positive region and expression intensity of osteogenic differentiation, and quantitative analysis was performed. RESULTS AND CONCLUSION:(1) The proliferation of bone marrow mesenchymal stem cells transfected with BMP2 and TGFβ2 was not statistically different from that of untransfected cells(P > 0.05).(2) The BMP2 and TGFβ2 mRNA levels were significantly higher in the transfected cells than untransfected ones(P < 0.05).(3) RUNX2 immunohistochemistry under the in vitro two-dimensional culture conditions and Vonkossa staining under the in vitro three-dimensional culture showed that the BMP2 and TGFβ2 co-transfected bone marrow mesenchymal stem cells had stronger osteogenic ability than BMP2 or TGFβ2 alone transfected cells(P < 0.05). In conclusion, the overexpression of recombinant plasmids of BMP2 and TGFβ2 carried by lentivirus synergistically promotes the osteogenic differentiation of rat bone marrow mesenchymal stem cells.
引文
[1]Li P, Bai Y, Yin G, et al. Synergistic and sequential effects of BMP-2, bFGF and VEGF on osteogenic differentiation of rat osteoblasts.J Bone Miner Metab. 2014;32(6):627-635.
[2]Barati D, Shariati SRP, Moeinzadeh S, et al. Spatiotemporal release of BMP-2 and VEGF enhances osteogenic and vasculogenic differentiation of human mesenchymal stem cells and endothelial colony-forming cells co-encapsulated in a patterned hydrogel.J Control Release. 2016;223:126-136.
[3]Nakano K, Murata K, Omokawa S, et al. Promotion of Osteogenesis and Angiogenesis in Vascularized Tissue-Engineered Bone Using Osteogenic Matrix Cell Sheets.Plast Reconstr Surg. 2016;137(5):1476-1484.
[4]Shen Y, Qiao H, Fan Q, et al. Potentiated Osteoinductivity via Cotransfection with BMP-2 and VEGF Genes in Microencapsulated C2C12 Cells.Biomed Res Int. 2015;2015:435253.
[5]Kim IS, Song YM, Hwang SJ.Osteogenic responses of human mesenchymal stromal cells to static stretch.J Dent Res. 2010;89(10):1129-1134.
[6]Taghiyar L, Hosseini S, Hesaraki M, et al. Isolation,Characterization and Osteogenic Potential of Mouse Digit Tip Blastema Cells in Comparison with Bone Marrow-Derived Mesenchymal Stem Cells In Vitro.Cell J. 2018;19(4):585-598.
[7]Bakopoulou A, Apatzidou D, Aggelidou E, et al. Isolation and prolonged expansion of oral mesenchymal stem cells under clinical-grade, GMP-compliant conditions differentially affects"stemness"properties.Stem Cell Res Ther. 2017;8(1):247.
[8]Shokohi R, Nabiuni M, Irian S, et al. In Vitro Effects of Wistar Rat Prenatal and Postnatal Cerebrospinal Fluid on Neural Differentiation and P roliferation of Mesenchymal Stromal Cells Derived from Bone Marrow.Cell J. 2018;19(4):537-544.
[9]Shi XL, Hu BB, Ren MM, et al. Hypoxia regulates the expression of OPG/RANKL mRNA in rat bone marrow mesenchymal stem cells.Shanghai Kou Qiang Yi Xue. 2017;26(3):258-262.
[10]Lin J, Shao J, Juan L, et al. Enhancing bone regeneration by combining mesenchymal stem cell sheets withβ-TCP/COL-I scaffolds.J Biomed Mater Res B Appl Biomater. 2018;106(5):2037-2045.
[11]He XT, Li X, Yin Y, et al. The effects of conditioned media generated by polarized macrophages on the cellular behaviours of bone marrow mesenchymal stem cells.J Cell Mol Med. 2018;22(2):1302-1315.
[12]Tirkkonen L, Haimi S, Huttunen S, et al. Osteogenic medium is superior to growth factors in differentiation of human adipose stem cells towards bone-forming cells in 3D culture.Eur Cell Mater. 2013;25:144-158.
[13]Jiang J, Fan CY, Zeng BF.Osteogenic differentiation effects on rat bone marrow-derived mesenchymal stromal cells by lentivirus-mediated co-transfection of human BMP2 gene and VEGF165 gene.Biotechnol Lett. 2008;30(2):197-203.
[14]Li H, Li J, Jiang J, et al. An osteogenesis/angiogenesis-stimulation artificial ligament for anterior cruciate ligament reconstruction.Acta Biomater. 2017;54:399-410.
[15]Perez RA, Kim JH, Buitrago JO, et al. Novel therapeutic core-shell hydrogel scaffolds with sequential delivery of cobalt and bone morphogenetic protein-2 for synergistic bone regeneration.Acta Biomater. 2015;23:295-308.
[16]Herath TDK, Larbi A, Teoh SH, et al. Neutrophil-mediated enhancement of angiogenesis and osteogenesis in a novel triple cell co-culture model with endothelial cells and osteoblasts.J Tissue Eng Regen Med. 2018;12(2):e1221-e1236.
[17]Liu S, Jin D, Wu JQ, et al. Neuropeptide Y stimulates osteoblastic differentiation and VEGF expression of bone marrow mesenchymal stem cells related to canonical Wnt signaling activating in vitro.Neuropeptides. 2016;56:105-113.
[18]Kumar S, Wan C, Ramaswamy G, et al. Mesenchymal stem cells expressing osteogenic and angiogenic factors synergistically enhance bone formation in a mouse model of segmental bone defect.Mol Ther. 2010;18(5):1026-1034.
[19]Fu TS, Chang YH, Wong CB, et al. Mesenchymal stem cells expressing baculovirus-engineered BMP-2 and VEGF enhance posterolateral spine fusion in a rabbit model.Spine J.2015;15(9):2036-2044.
[20]Wang W, Kratz K, Behl M, et al. The interaction of adipose-derived human mesenchymal stem cells and polyether ether ketone.Clin Hemorheol Microcirc. 2015;61(2):301-321.
[21]Han TY, Liu XW, Liang N, et al. In vitro effects of recombinant adenovirus-mediated bone morphogenetic protein 2/vascular endothelial growth factor 165 on osteogenic differentiation of bone marrow mesenchymal stem cells.Artif Cells Nanomed Biotechnol. 2017;45(1):108-114.
[22]Lin Z, Wang JS, Lin L, et al. Effects of BMP2 and VEGF165on the osteogenic differentiation of rat bone marrow-derived mesenchymal stem cells.Exp Ther Med. 2014;7(3):625-629.
[23]Kanczler JM, Ginty PJ, White L, et al. The effect of the delivery of vascular endothelial growth factor and bone morphogenic protein-2 to osteoprogenitor cell populations on bone formation.Biomaterials. 2010;31(6):1242-1250.
[24]Tian XB, Sun L, Yang SH, et al. Ectopic osteogenesis of mouse bone marrow stromal cells transfected with BMP2/VEGF(165)genes in vivo.Orthop Surg. 2009;1(4):322-325.
[25]K?rner E, B?ckesj?CM, Cedervall J, et al. Dynamics of gene expression during bone matrix formation in osteogenic cultures derived from human embryonic stem cells in vitro.Biochim Biophys Acta. 2009;1790(2):110-118.
[26]Janko M, Sahm J, Schaible A, et al. Comparison of three different types of scaffolds preseeded with human bone marrow mononuclear cells on the bone healing in a femoral critical size defect model of the athymic rat.J Tissue Eng Regen Med. 2018;12(3):653-666.
[27]Farhadi J, Jaquiery C, Barbero A, et al.Differentiation-dependent up-regulation of BMP-2, TGF-beta1,and VEGF expression by FGF-2 in human bone marrow stromal cells.Plast Reconstr Surg. 2005;116(5):1379-1386.
[28]Curtin CM, Tierney EG, McSorley K, et al. Combinatorial gene therapy accelerates bone regeneration:non-viral dual delivery of VEGF and BMP2 in a collagen-nanohydroxyapatite scaffold.Adv Healthc Mater.2015;4(2):223-227.
[29]Xiao C, Zhou H, Liu G, et al. Bone marrow stromal cells with a combined expression of BMP-2 and VEGF-165 enhanced bone regeneration.Biomed Mater. 2011;6(1):015013.
[30]Song X, Liu S, Qu X, et al. BMP2 and VEGF promote angiogenesis but retard terminal differentiation of osteoblasts in bone regeneration by up-regulating Id1.Acta Biochim Biophys Sin(Shanghai). 2011;43(10):796-804.
[31]Lin CY, Chang YH, Kao CY, et al. Augmented healing of critical-size calvarial defects by baculovirus-engineered MSCs that persistently express growth factors.Biomaterials.2012;33(14):3682-3692.
[32]Zhang C, Yu L, Liu S, et al. Human amnion-derived mesenchymal stem cells promote osteogenic and angiogenic differentiation of human adipose-derived stem cells.PLoS One. 2017;12(10):e0186253.
[33]胡正雄,李彪,蓝天.TGF-β2和geneX对BrdU标记骨髓间充质干细胞增殖与成骨分化的作用[J].昆明医科大学学报, 2016,37(2):10-14.
[34]张爱军,闫志勇.TGF-β对创伤愈合与瘢痕形成的影响及中药的干预作用[J].西北药学杂志,2013,16(1):101-105.
[2]Barati D, Shariati SRP, Moeinzadeh S, et al. Spatiotemporal release of BMP-2 and VEGF enhances osteogenic and vasculogenic differentiation of human mesenchymal stem cells and endothelial colony-forming cells co-encapsulated in a patterned hydrogel.J Control Release. 2016;223:126-136.
[3]Nakano K, Murata K, Omokawa S, et al. Promotion of Osteogenesis and Angiogenesis in Vascularized Tissue-Engineered Bone Using Osteogenic Matrix Cell Sheets.Plast Reconstr Surg. 2016;137(5):1476-1484.
[4]Shen Y, Qiao H, Fan Q, et al. Potentiated Osteoinductivity via Cotransfection with BMP-2 and VEGF Genes in Microencapsulated C2C12 Cells.Biomed Res Int. 2015;2015:435253.
[5]Kim IS, Song YM, Hwang SJ.Osteogenic responses of human mesenchymal stromal cells to static stretch.J Dent Res. 2010;89(10):1129-1134.
[6]Taghiyar L, Hosseini S, Hesaraki M, et al. Isolation,Characterization and Osteogenic Potential of Mouse Digit Tip Blastema Cells in Comparison with Bone Marrow-Derived Mesenchymal Stem Cells In Vitro.Cell J. 2018;19(4):585-598.
[7]Bakopoulou A, Apatzidou D, Aggelidou E, et al. Isolation and prolonged expansion of oral mesenchymal stem cells under clinical-grade, GMP-compliant conditions differentially affects"stemness"properties.Stem Cell Res Ther. 2017;8(1):247.
[8]Shokohi R, Nabiuni M, Irian S, et al. In Vitro Effects of Wistar Rat Prenatal and Postnatal Cerebrospinal Fluid on Neural Differentiation and P roliferation of Mesenchymal Stromal Cells Derived from Bone Marrow.Cell J. 2018;19(4):537-544.
[9]Shi XL, Hu BB, Ren MM, et al. Hypoxia regulates the expression of OPG/RANKL mRNA in rat bone marrow mesenchymal stem cells.Shanghai Kou Qiang Yi Xue. 2017;26(3):258-262.
[10]Lin J, Shao J, Juan L, et al. Enhancing bone regeneration by combining mesenchymal stem cell sheets withβ-TCP/COL-I scaffolds.J Biomed Mater Res B Appl Biomater. 2018;106(5):2037-2045.
[11]He XT, Li X, Yin Y, et al. The effects of conditioned media generated by polarized macrophages on the cellular behaviours of bone marrow mesenchymal stem cells.J Cell Mol Med. 2018;22(2):1302-1315.
[12]Tirkkonen L, Haimi S, Huttunen S, et al. Osteogenic medium is superior to growth factors in differentiation of human adipose stem cells towards bone-forming cells in 3D culture.Eur Cell Mater. 2013;25:144-158.
[13]Jiang J, Fan CY, Zeng BF.Osteogenic differentiation effects on rat bone marrow-derived mesenchymal stromal cells by lentivirus-mediated co-transfection of human BMP2 gene and VEGF165 gene.Biotechnol Lett. 2008;30(2):197-203.
[14]Li H, Li J, Jiang J, et al. An osteogenesis/angiogenesis-stimulation artificial ligament for anterior cruciate ligament reconstruction.Acta Biomater. 2017;54:399-410.
[15]Perez RA, Kim JH, Buitrago JO, et al. Novel therapeutic core-shell hydrogel scaffolds with sequential delivery of cobalt and bone morphogenetic protein-2 for synergistic bone regeneration.Acta Biomater. 2015;23:295-308.
[16]Herath TDK, Larbi A, Teoh SH, et al. Neutrophil-mediated enhancement of angiogenesis and osteogenesis in a novel triple cell co-culture model with endothelial cells and osteoblasts.J Tissue Eng Regen Med. 2018;12(2):e1221-e1236.
[17]Liu S, Jin D, Wu JQ, et al. Neuropeptide Y stimulates osteoblastic differentiation and VEGF expression of bone marrow mesenchymal stem cells related to canonical Wnt signaling activating in vitro.Neuropeptides. 2016;56:105-113.
[18]Kumar S, Wan C, Ramaswamy G, et al. Mesenchymal stem cells expressing osteogenic and angiogenic factors synergistically enhance bone formation in a mouse model of segmental bone defect.Mol Ther. 2010;18(5):1026-1034.
[19]Fu TS, Chang YH, Wong CB, et al. Mesenchymal stem cells expressing baculovirus-engineered BMP-2 and VEGF enhance posterolateral spine fusion in a rabbit model.Spine J.2015;15(9):2036-2044.
[20]Wang W, Kratz K, Behl M, et al. The interaction of adipose-derived human mesenchymal stem cells and polyether ether ketone.Clin Hemorheol Microcirc. 2015;61(2):301-321.
[21]Han TY, Liu XW, Liang N, et al. In vitro effects of recombinant adenovirus-mediated bone morphogenetic protein 2/vascular endothelial growth factor 165 on osteogenic differentiation of bone marrow mesenchymal stem cells.Artif Cells Nanomed Biotechnol. 2017;45(1):108-114.
[22]Lin Z, Wang JS, Lin L, et al. Effects of BMP2 and VEGF165on the osteogenic differentiation of rat bone marrow-derived mesenchymal stem cells.Exp Ther Med. 2014;7(3):625-629.
[23]Kanczler JM, Ginty PJ, White L, et al. The effect of the delivery of vascular endothelial growth factor and bone morphogenic protein-2 to osteoprogenitor cell populations on bone formation.Biomaterials. 2010;31(6):1242-1250.
[24]Tian XB, Sun L, Yang SH, et al. Ectopic osteogenesis of mouse bone marrow stromal cells transfected with BMP2/VEGF(165)genes in vivo.Orthop Surg. 2009;1(4):322-325.
[25]K?rner E, B?ckesj?CM, Cedervall J, et al. Dynamics of gene expression during bone matrix formation in osteogenic cultures derived from human embryonic stem cells in vitro.Biochim Biophys Acta. 2009;1790(2):110-118.
[26]Janko M, Sahm J, Schaible A, et al. Comparison of three different types of scaffolds preseeded with human bone marrow mononuclear cells on the bone healing in a femoral critical size defect model of the athymic rat.J Tissue Eng Regen Med. 2018;12(3):653-666.
[27]Farhadi J, Jaquiery C, Barbero A, et al.Differentiation-dependent up-regulation of BMP-2, TGF-beta1,and VEGF expression by FGF-2 in human bone marrow stromal cells.Plast Reconstr Surg. 2005;116(5):1379-1386.
[28]Curtin CM, Tierney EG, McSorley K, et al. Combinatorial gene therapy accelerates bone regeneration:non-viral dual delivery of VEGF and BMP2 in a collagen-nanohydroxyapatite scaffold.Adv Healthc Mater.2015;4(2):223-227.
[29]Xiao C, Zhou H, Liu G, et al. Bone marrow stromal cells with a combined expression of BMP-2 and VEGF-165 enhanced bone regeneration.Biomed Mater. 2011;6(1):015013.
[30]Song X, Liu S, Qu X, et al. BMP2 and VEGF promote angiogenesis but retard terminal differentiation of osteoblasts in bone regeneration by up-regulating Id1.Acta Biochim Biophys Sin(Shanghai). 2011;43(10):796-804.
[31]Lin CY, Chang YH, Kao CY, et al. Augmented healing of critical-size calvarial defects by baculovirus-engineered MSCs that persistently express growth factors.Biomaterials.2012;33(14):3682-3692.
[32]Zhang C, Yu L, Liu S, et al. Human amnion-derived mesenchymal stem cells promote osteogenic and angiogenic differentiation of human adipose-derived stem cells.PLoS One. 2017;12(10):e0186253.
[33]胡正雄,李彪,蓝天.TGF-β2和geneX对BrdU标记骨髓间充质干细胞增殖与成骨分化的作用[J].昆明医科大学学报, 2016,37(2):10-14.
[34]张爱军,闫志勇.TGF-β对创伤愈合与瘢痕形成的影响及中药的干预作用[J].西北药学杂志,2013,16(1):101-105.