慢病毒介导bFGF和BMP-2双基因转染对兔骨髓间充质干细胞增殖的影响
|
摘要
目的构建携带骨形成蛋白-2(BMP-2)与碱性成纤维细胞生长因子(bFGF)双基因过表达的慢病毒载体,观察其对兔骨髓间充质干细胞(BMSCs)增殖的影响。方法体外培养兔BMSCs,构建携带b FGF和BMP-2双基因的重组慢病毒载体,转染至BMSCs诱导两种基因过表达;流式细胞仪检测转染效率;Western blot方法验证双基因转染模型构建成功;MTT方法检测细胞增殖水平。结果慢病毒以最佳感染复数(MOI)100转染BMSCs时转染效率最佳,Western blot检测到转染后细胞bFGF和BMP-2蛋白表达量明显比对照增加(P<0.05);MTT结果发现,过表达bFGF和BMP-2的BMSCs细胞的增殖水平增加(P<0.05)。结论 bFGF和BMP-2双基因共表达重组腺病毒载体促进BMSCs细胞增殖。
Objective To construct bone morphogenetic protein 2(BMP-2) and basic fibroblast growth factor(bFGF) double gene lentivirus vectors, and to observe its effect on the proliferation of bone marrow mesenchymal stem cells(BMSCs) in rabbit. Methods Rabbit BMSCs were cultured in vitro. The recombinant lentivirus vectors carrying b FGF and BMP-2 were constructed and transfected into BMSCs to induce overexpression of two genes. The transfection efficiency was detected by flow cytometry. Western blot was used to verify the success of the two-gene transfection model. The cell proliferation was detected by MTT. Results Lentivirus was transfected with the optimal complex number(MOI) 100 for BMSCs. The expression level of bFGF and BMP-2 proteins was significantly increased in transfected cells than in the control group(P<0.05). MTT results showed that the proliferation rates of BMSCs with overexpression of bFGF and BMP-2 increased(P<0.05). Conclusion The combined b FGF and BMP-2 recombinant adenovirus vector promoted the proliferation of BMSCs cells.
Objective To construct bone morphogenetic protein 2(BMP-2) and basic fibroblast growth factor(bFGF) double gene lentivirus vectors, and to observe its effect on the proliferation of bone marrow mesenchymal stem cells(BMSCs) in rabbit. Methods Rabbit BMSCs were cultured in vitro. The recombinant lentivirus vectors carrying b FGF and BMP-2 were constructed and transfected into BMSCs to induce overexpression of two genes. The transfection efficiency was detected by flow cytometry. Western blot was used to verify the success of the two-gene transfection model. The cell proliferation was detected by MTT. Results Lentivirus was transfected with the optimal complex number(MOI) 100 for BMSCs. The expression level of bFGF and BMP-2 proteins was significantly increased in transfected cells than in the control group(P<0.05). MTT results showed that the proliferation rates of BMSCs with overexpression of bFGF and BMP-2 increased(P<0.05). Conclusion The combined b FGF and BMP-2 recombinant adenovirus vector promoted the proliferation of BMSCs cells.
引文
[1]Jin YZ,Lee JH.Mesenchymal stem cell therapy for bone regeneration[J].Clin Orthop Surg,2018,10(3):271-278.
[2]Krebsbach PH,Kuznetsov SA,Bianco P,et al.Bone marrow stromal cells:characterization and clinical application[J].Crit Rev Oral Biol Med,1999,10(2):165-181.
[3]Han W,Yu Y,Liu XY.Local signals in stem cell-based bone marrow regeneration[J].Cell Res,2006,16(2):189-195.
[4]Bianco P."Mesenchymal"stem cells[J].Annual Review of Cell and Developmental Biology,2014,30(1):677-704.
[5]Hsieh MK,Wu CJ,Chen CC,et al.BMP-2 gene transfection of bone marrow stromal cells to induce osteoblastic differentiation in a rat calvarial defect model[J].Mater Sci Eng C Mater Biol Appl,2018,91:806-816.
[6]Shu Y,Yu Y,Zhang S,et al.The immunomodulatory role of sulfated chitosan in BMP-2-mediated bone regeneration[J].Biomater Sci,2018,6(9):2496-2507.
[7]Marie PJ,Miraoui H,Severe N.FGF/FGFR signaling in bone formation:progress and perspectives[J].Growth Factors,2012,30(2):117-123.
[8]Zhang F,Peng WX,Wang L,et al.Role of FGF-2 transfected bone marrow mesenchymal stem cells in engineered bone tissue for repair of a vascular necrosis of femoral head in rabbits[J].Cell Physiol Biochem,2018,48(2):773-784.
[9]Byers BA,Guldberg RE,García AJ.Synergy between genetic and tissue engineering:Runx2 overexpression and in vitro construct development enhance in vivo mineralization[J].Tissue Eng,2004,10(11-12):1757-1766.
[10]Yang JH,Kim HJ,Kim SE,et al.The effect of bone morphogenic protein-2-coated tri-calcium phosphate/hydroxyapatite on new bone formation in a rat model of femoral distraction osteogenesis[J].Cytotherapy,2012,14(3):315-326.
[11]Wu S,Xiao Z,Song J,et al.Evaluation of BMP-2 enhances the osteoblast differentiation of human amnion mesenchymal stem cells seeded on Nano-Hydroxyapatite/Collagen/Poly(l-Lactide)[J].Int JMol Sci,2018,19(8):pii:E2171.
[12]Feng L,Li Y,Zeng W,et al.Enhancing effects of basic fibroblast growth factor and fibronectin on osteoblast adhesion to bone scaffolds for bone tissue engineering through extracellular matrixintegrin pathway[J].Exp Ther Med,2017,14(6):6087-6092.
[13]Du M,Zhu T,Duan X,et al.Acellular dermal matrix loading with bFGF achieves similar acceleration of bone regeneration to BMP-2via differential effects on recruitment,proliferation and sustained osteodifferentiation of mesenchymal stem cells[J].Mater Sci Eng CMater Biol Appl,2017,70(Pt 1):62-70.
[14]Mura M,Pisano F,Malpasso G,et al.Novel IRES-based lentivirus co-expressing IGF1 and BMP2 enhances both cardiomyogenesis and cytoprotection of bone marrow-derived mesenchymal stem cells[J].Eur Heart J,2013,34(suppl 1):147.
[2]Krebsbach PH,Kuznetsov SA,Bianco P,et al.Bone marrow stromal cells:characterization and clinical application[J].Crit Rev Oral Biol Med,1999,10(2):165-181.
[3]Han W,Yu Y,Liu XY.Local signals in stem cell-based bone marrow regeneration[J].Cell Res,2006,16(2):189-195.
[4]Bianco P."Mesenchymal"stem cells[J].Annual Review of Cell and Developmental Biology,2014,30(1):677-704.
[5]Hsieh MK,Wu CJ,Chen CC,et al.BMP-2 gene transfection of bone marrow stromal cells to induce osteoblastic differentiation in a rat calvarial defect model[J].Mater Sci Eng C Mater Biol Appl,2018,91:806-816.
[6]Shu Y,Yu Y,Zhang S,et al.The immunomodulatory role of sulfated chitosan in BMP-2-mediated bone regeneration[J].Biomater Sci,2018,6(9):2496-2507.
[7]Marie PJ,Miraoui H,Severe N.FGF/FGFR signaling in bone formation:progress and perspectives[J].Growth Factors,2012,30(2):117-123.
[8]Zhang F,Peng WX,Wang L,et al.Role of FGF-2 transfected bone marrow mesenchymal stem cells in engineered bone tissue for repair of a vascular necrosis of femoral head in rabbits[J].Cell Physiol Biochem,2018,48(2):773-784.
[9]Byers BA,Guldberg RE,García AJ.Synergy between genetic and tissue engineering:Runx2 overexpression and in vitro construct development enhance in vivo mineralization[J].Tissue Eng,2004,10(11-12):1757-1766.
[10]Yang JH,Kim HJ,Kim SE,et al.The effect of bone morphogenic protein-2-coated tri-calcium phosphate/hydroxyapatite on new bone formation in a rat model of femoral distraction osteogenesis[J].Cytotherapy,2012,14(3):315-326.
[11]Wu S,Xiao Z,Song J,et al.Evaluation of BMP-2 enhances the osteoblast differentiation of human amnion mesenchymal stem cells seeded on Nano-Hydroxyapatite/Collagen/Poly(l-Lactide)[J].Int JMol Sci,2018,19(8):pii:E2171.
[12]Feng L,Li Y,Zeng W,et al.Enhancing effects of basic fibroblast growth factor and fibronectin on osteoblast adhesion to bone scaffolds for bone tissue engineering through extracellular matrixintegrin pathway[J].Exp Ther Med,2017,14(6):6087-6092.
[13]Du M,Zhu T,Duan X,et al.Acellular dermal matrix loading with bFGF achieves similar acceleration of bone regeneration to BMP-2via differential effects on recruitment,proliferation and sustained osteodifferentiation of mesenchymal stem cells[J].Mater Sci Eng CMater Biol Appl,2017,70(Pt 1):62-70.
[14]Mura M,Pisano F,Malpasso G,et al.Novel IRES-based lentivirus co-expressing IGF1 and BMP2 enhances both cardiomyogenesis and cytoprotection of bone marrow-derived mesenchymal stem cells[J].Eur Heart J,2013,34(suppl 1):147.
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |