不同状态BMP-2对体外EMSCs成骨分化的作用
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摘要
[目的]探讨骨形成蛋白(BMP-2)与组织型谷氨酰胺转氨酶(tTG, TG2)交联形成固化型和BMP-2游离型细胞生长因子,两种不同状态的BMP-2对鼻粘膜间充质干细胞(EMSCs)向成骨细胞分化的影响;[方法]体外培养、扩增并鉴定EMSCs;实验分四组:TG2+BMP-2、BMP-2、TG2和空白对照;四组细胞均采用定向成骨诱导培养基培养7 d;检测各组碱性磷酸酶活性及形成的钙结节面积大小;免疫印迹法检测成骨诱导相关蛋白表达水平;MTT法检测TG2、BMP-2和TG2+BMP-2对EMSCs增殖的影响。[结果]成骨诱导7 d后,TG2+BMP-2组细胞碱性磷酸酶活性较高,形成的钙结节较大,骨相关蛋白表达水平较高;TG2+BMP-2,TG2和BMP-2对EMSCs增殖能力无明显影响。[结论] TG2+BMP-2交联固化型细胞生长因子对EMSCs向成骨细胞分化表现出较强的促进作用,是组织工程化骨中理想的诱导成骨的细胞因子,具有广阔的应用前景。
[Objective]To explore the role of bone morphogenetic protein-2(BMP-2) in two forms,the combination of BMP-2 with tissue transglutaminase(TG2) by cross-linking and free BMP-2,on osteogenic differentiation of ectomesenchymal stem cells(EMSCs) in vitro.[Methods]The EMSCs,isolated from nasal mucosa of rat,then expanded and identified in vitro,were divided into 4 groups,including TG2+BMP-2,BMP-2,TG2,and blank control.The cells were further cultured under the 4 conditions in osteogenic induction medium for 7 days.The cell proliferation were detected by MTT assay,while the activity of alkaline phosphatase and the area of calcium nodules formed were detected by chemical staining methods,and the expression level of osteogenic induced proteins was detected by Western blotting.[Results]After 7 days of osteogenic induction culture,although no significant differences in cell proliferation among the 4 groups(P>0.05),the TG2+BMP-2 group presented significantly higher alkaline phosphatase activity,associated with larger calcium nodules and higher expression of bone-related proteins than the other 3 group(P<0.05).[Conclusion]The combination of TG2 with BMP-2 shows improved osteoinductive effect on EMSCs,which might be an ideal osteoinductive cytokine for engineered bones.
[Objective]To explore the role of bone morphogenetic protein-2(BMP-2) in two forms,the combination of BMP-2 with tissue transglutaminase(TG2) by cross-linking and free BMP-2,on osteogenic differentiation of ectomesenchymal stem cells(EMSCs) in vitro.[Methods]The EMSCs,isolated from nasal mucosa of rat,then expanded and identified in vitro,were divided into 4 groups,including TG2+BMP-2,BMP-2,TG2,and blank control.The cells were further cultured under the 4 conditions in osteogenic induction medium for 7 days.The cell proliferation were detected by MTT assay,while the activity of alkaline phosphatase and the area of calcium nodules formed were detected by chemical staining methods,and the expression level of osteogenic induced proteins was detected by Western blotting.[Results]After 7 days of osteogenic induction culture,although no significant differences in cell proliferation among the 4 groups(P>0.05),the TG2+BMP-2 group presented significantly higher alkaline phosphatase activity,associated with larger calcium nodules and higher expression of bone-related proteins than the other 3 group(P<0.05).[Conclusion]The combination of TG2 with BMP-2 shows improved osteoinductive effect on EMSCs,which might be an ideal osteoinductive cytokine for engineered bones.
引文
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[8]Chen Z,Zuo X,Li H,et al.Effects of melatonin on maturation,histone acetylation,autophagy of porcine oocytes and subsequent embryonic development[J].Animal Sci J,2017,88(9):1298-1310.
[9]Nakagawa Y,Ikeda K,Akakabe Y,et al.Paracrine osteogenic signals via bone morphogenetic protein-2 accelerate the atherosclerotic intimal calcification in vivo[J].Arteriosclerosis Thrombosis Vascular Biology,2010,30(10):1908-1915.
[10]Yin X,Chen Z,Liu Z,et al.Tissue transglutaminase(TG2)activity regulates osteoblast differentiation and mineralization in the SAOS-2 cell line[J].Brazilian J Med Biol Res,2012,45(8):693-700.
[11]Chen Q,Zhang Z,Liu J,et al.A fibrin matrix promotes the differentiation of EMSCs isolated from nasal respiratory mucosa to myelinating phenotypical Schwann-like cells[J].Molecules Cells,2015,38(3):221.
[12]Chang YP,Hong HP,Lee YH,et al.The canine epiphyseal-derived mesenchymal stem cells are comparable to bone marrow derived-mesenchymal stem cells[J].J Veterinary Med Sci,2015,77(3):273-280.
[13]Otto A,Oliver H,Jane M.A method for the rapid determination of alkaline phosphatase with five cubic millimeters of serum[J].J Biol Chemi,1946,164:321-329.
[14]Bensimon-Brito A,Cardeira J,Dionísio G,et al.Revisiting in vivo staining with alizarin red S-a valuable approach to analyse zebrafish skeletal mineralization during development and regeneration[J].BMC Develop Biol,2016,16(1):2.
[15]Fratzl P.Collagen:structure and mechanics,an introduction[M]//Boston:Collagen.Springer,2008:1-13.
[16]Ducy P,Desbois C,Boyce B,et al.Increased bone formation in osteocalcin-deficient mice[J].Nature,1996,382(6590):448.
[17]Rodrigues AM,Caetano-Lopes J,Vale AC,et al.Low osteocalcin/collagen type I bone gene expression ratio is associated with hip fragility fractures[J].Bone,2012,51(6):981-989.
[18]Niswander L,Martin GR.FGF-4 and BMP-2 have opposite effects on limb growth[J].Nature,1993,361(6407):68.2018-05-24
[2]马武秀,程迅生.自体骨移植修复骨缺损的研究进展[J].中国骨与关节损伤杂志,2011,26(6):574-576.
[3]雷建平,徐昊,张克良.rhBMP-2骨修复材料在后路腰椎内固定植骨融合术中应用[J].中国矫形外科杂志,2018,26(5):390-394.
[4]Zhang Z,He Q,Deng W,et al.Nasal ectomesenchymal stem cells:multi-lineage differentiation and transformation effects on fibrin gels[J].Biomaterials,2015,49(1):57-67.
[5]张志坚,刘锦波,姜平,等.大鼠嗅粘膜原代培养细胞的分类及细胞社会学特性研究[J].江苏大学学报(医学版),2004(1):4-7,96.
[6]Chen DI,Zhao M,Mundy GR.Bone morphogenetic proteins[J].Growth factors,2004,22(4):233-241.
[7]Goumans MJ,Zwijsen A,Ten Dijke P,et al.Bone morphogenetic proteins in vascular homeostasis and disease[J].Cold Spring Harbor Perspectives Biology,2018,10(2):a031989.
[8]Chen Z,Zuo X,Li H,et al.Effects of melatonin on maturation,histone acetylation,autophagy of porcine oocytes and subsequent embryonic development[J].Animal Sci J,2017,88(9):1298-1310.
[9]Nakagawa Y,Ikeda K,Akakabe Y,et al.Paracrine osteogenic signals via bone morphogenetic protein-2 accelerate the atherosclerotic intimal calcification in vivo[J].Arteriosclerosis Thrombosis Vascular Biology,2010,30(10):1908-1915.
[10]Yin X,Chen Z,Liu Z,et al.Tissue transglutaminase(TG2)activity regulates osteoblast differentiation and mineralization in the SAOS-2 cell line[J].Brazilian J Med Biol Res,2012,45(8):693-700.
[11]Chen Q,Zhang Z,Liu J,et al.A fibrin matrix promotes the differentiation of EMSCs isolated from nasal respiratory mucosa to myelinating phenotypical Schwann-like cells[J].Molecules Cells,2015,38(3):221.
[12]Chang YP,Hong HP,Lee YH,et al.The canine epiphyseal-derived mesenchymal stem cells are comparable to bone marrow derived-mesenchymal stem cells[J].J Veterinary Med Sci,2015,77(3):273-280.
[13]Otto A,Oliver H,Jane M.A method for the rapid determination of alkaline phosphatase with five cubic millimeters of serum[J].J Biol Chemi,1946,164:321-329.
[14]Bensimon-Brito A,Cardeira J,Dionísio G,et al.Revisiting in vivo staining with alizarin red S-a valuable approach to analyse zebrafish skeletal mineralization during development and regeneration[J].BMC Develop Biol,2016,16(1):2.
[15]Fratzl P.Collagen:structure and mechanics,an introduction[M]//Boston:Collagen.Springer,2008:1-13.
[16]Ducy P,Desbois C,Boyce B,et al.Increased bone formation in osteocalcin-deficient mice[J].Nature,1996,382(6590):448.
[17]Rodrigues AM,Caetano-Lopes J,Vale AC,et al.Low osteocalcin/collagen type I bone gene expression ratio is associated with hip fragility fractures[J].Bone,2012,51(6):981-989.
[18]Niswander L,Martin GR.FGF-4 and BMP-2 have opposite effects on limb growth[J].Nature,1993,361(6407):68.2018-05-24