骨形成蛋白-2与转化生长因子-β_3对牙髓干细胞成骨向分化的对比研究
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摘要
目的探讨转化生长因子-β_3(transforming growth factor-β_3,TGF-β_3)、骨形成蛋白-2(bone morphogenetic protein-2,BMP-2)对牙髓干细胞(dental pulp stem cells,DPSCs)成骨向分化的影响。方法健康新西兰幼兔4只,取牙髓组织采用酶解组织块法分离培养获得DPSCs,镜下观察细胞形态及生长状况。将培养的第3代DPSCs分为BMP-2组和TGF-β_3组,分别加入浓度为80μg/L的BMP-2、TGF-β_3的完全培养液,采用免疫组织化学法于诱导第3、7天检测Ⅰ型胶原蛋白(collagen-Ⅰ,COL-Ⅰ)表达,于第7、14天检测骨钙素(osteocalcin,OC)表达;诱导第7、14天,对BMP-2组、TGF-β_3组行碱性磷酸酶(alkaline phosphatase,ALP)染色后,倒置显微镜下观察ALP活性染色结果。结果兔DPSCs分离、培养48h,组织块周围有细胞游出、贴壁生长,培养的第3代兔DPSCs大多为长梭形,也可为多角形,呈巢式或集落生长;TGF-β_3组诱导第3天COL-1表达的累计吸光度(integrated optiacal density,IOD)值(159 676.79±63 959.56)高于BMP-2组(44 047.33±15 853.65)(P<0.05),诱导第7天COL-1表达的IOD值(245 378.26±60 084.60)与BMP-2组(223 294.43±80 266.72)比较差异无统计学意义(P>0.05);TGF-β_3组诱导第7天OC表达的IOD值(109 568.82±14 079.88)高于BMP-2组(55 168.57±1 694.93)(P<0.05),诱导第14天OC表达的IOD值(411 544.95±82 803.24)与BMP-2组(360 103.54±20 835.36)比较差异无统计学意义(P>0.05);BMP-2组、TGF-β_3组诱导第7天出现少量淡蓝色ALP阳性区,诱导第14天蓝色ALP阳性区增大且蓝染颜色加重,镜下观察2组无明显差异。结论与BMP-2比较,TGF-β_3对早期兔DPSCs向成骨细胞分化有较明显的促进作用。
Objective To investigate the effects of transforming growth factor-β_3(TGF-β_3)and bone morphogenetic protein-2(BMP-2)on osteogenic differentiation of dental pulp stem cells(DPSCs).Methods The pulp tissues from 4 healthy New Zealand rabbits were isolated and cultured by enzymolysis tissue mass method to obtain DPSCs.The morphology and growth of DPSCs were observed under microscope.The third passage DPSCs were divided into BMP-2 group and TGF-β_3 group,and added with 80μg/L BMP-2 and TGF-β_3 culture solution,respectively.The expressions of collagen-Ⅰ(COL-Ⅰ)on day 3and 7 of culturing and osteocalcin(OC)in osteoblasts on day 7and 14 of culturing were detected by immunohistochemical method.ALP activity was observed by inverted microscope on day 7 and 14 after ALP staining in BMP-2group and TGF-β_3 group.Results After 48 hof isolating and culturing,the cells swam from the tissue block and the cells grew well.Most of the cultured DPSCs were in fusiform or polygonal.On day 3 of induction,integrated optical density(IOD)value of COL-Ⅰ was significantly higher in TGF-β_3 group(159 676.79±63 959.56)than that in BMP-2 group(44 047.33±15 853.65)(P<0.05).There was no significant difference in the expression of IOD between TGF-β_3 group(245 378.26±60 084.60)and BMP-2 group(223 294.43±80 266.72)on day 7 of induction(P>0.05).The IOD value of OC was significantly higher in TGF-β_3 group(109 568.82±14 079.88)than that in BMP-2 group(55 168.57±1 694.93)on day 7 of induction(P<0.05),and there was no significant difference between TGF-β_3 group(411 544.95±82 803.24)and BMP-2 group(360 103.54±20 835.36)on day 14 of induction(P>0.05).On day 7 of induction,a small amount of light blue ALP positive areas appeared in both groups,which increased in area and deepened in color on day 14 of induction,showing no significant difference in ALP staining between two groups.Conclusion Compared with BMP-2,TGF-β_3 is more effective to promote the osteogenic differentiation of rabbit DPSCs in early stage.
Objective To investigate the effects of transforming growth factor-β_3(TGF-β_3)and bone morphogenetic protein-2(BMP-2)on osteogenic differentiation of dental pulp stem cells(DPSCs).Methods The pulp tissues from 4 healthy New Zealand rabbits were isolated and cultured by enzymolysis tissue mass method to obtain DPSCs.The morphology and growth of DPSCs were observed under microscope.The third passage DPSCs were divided into BMP-2 group and TGF-β_3 group,and added with 80μg/L BMP-2 and TGF-β_3 culture solution,respectively.The expressions of collagen-Ⅰ(COL-Ⅰ)on day 3and 7 of culturing and osteocalcin(OC)in osteoblasts on day 7and 14 of culturing were detected by immunohistochemical method.ALP activity was observed by inverted microscope on day 7 and 14 after ALP staining in BMP-2group and TGF-β_3 group.Results After 48 hof isolating and culturing,the cells swam from the tissue block and the cells grew well.Most of the cultured DPSCs were in fusiform or polygonal.On day 3 of induction,integrated optical density(IOD)value of COL-Ⅰ was significantly higher in TGF-β_3 group(159 676.79±63 959.56)than that in BMP-2 group(44 047.33±15 853.65)(P<0.05).There was no significant difference in the expression of IOD between TGF-β_3 group(245 378.26±60 084.60)and BMP-2 group(223 294.43±80 266.72)on day 7 of induction(P>0.05).The IOD value of OC was significantly higher in TGF-β_3 group(109 568.82±14 079.88)than that in BMP-2 group(55 168.57±1 694.93)on day 7 of induction(P<0.05),and there was no significant difference between TGF-β_3 group(411 544.95±82 803.24)and BMP-2 group(360 103.54±20 835.36)on day 14 of induction(P>0.05).On day 7 of induction,a small amount of light blue ALP positive areas appeared in both groups,which increased in area and deepened in color on day 14 of induction,showing no significant difference in ALP staining between two groups.Conclusion Compared with BMP-2,TGF-β_3 is more effective to promote the osteogenic differentiation of rabbit DPSCs in early stage.
引文
[1]RODRIGUEZ-LOZANO F J,BUENO C,INSAUSTI C L,et al.Mesenchymal stem cells derived from dental tissues[J].Int Endod J,2011,44(9):800-806.
[2]LIU J,YU F,SUN Y,et al.Concise reviews:characteristics and potential applications of human dental tissue-derived mesenchymal stem cells[J].Stem Cells,2015,33(3):627-638.
[3]DOLE N S,MAZUR C M,ACEVEDO C,et al.Osteocyteintrinsic TGF-βsignaling regulates bone quality through perilacunar/canalicular remodeling[J].Cell Rep,2017,21(9):2585-2596.
[4]刘晓文,木合塔尔·霍加.转化生长因子-β3对兔牙髓干细胞增殖及骨向分化的影响[J].中华实用诊断与治疗杂志,2016,30(3):242-245.
[5]JOHNSEN S A,SUBRAMANIAM M,KATAGIRI T,et al.Transcriptional regulation of Smad2is required for enhancement of TGF-β/Smad signaling by TGF-βinducible early gene[J].JCell Biochem,2002,87(2):233-241.
[6]王腾,木合塔尔·霍加.外源性转化生长因子-β3联合兔牙髓干细胞对兔骨缺损处成骨细胞转化生长因子-β3表达的影响[J].中华实用诊断与治疗杂志,2017,31(6):525-529.
[7]BEEDERMAN M,LAMPLOT J D,NAN G,et al.BMPsignaling in mesenchymal stem cell differentiation and bone formation[J].J Biomed Sci Eng,2013,6(8):32-52.
[8]JUNG U W,LEE I K,PARK J Y,et al.The efficacy of BMP-2preloaded on bone substitute or hydrogel for bone regeneration at peri-implant defects in dogs[J].Clin Oral Implants Res,2015,26(12):1456-1465.
[9]TOOM A,AREND A,GUNNARSSON D,et al.Bone formation zones in heterotopic ossifications:histologic findings and increased expression of bone morphogenetic protein 2and transforming growth factors-β2 andβ3[J].Calcif Tissue Int,2007,80(4):259-267.
[10]CHEAH F S,WINKLER C,JABS E W,et al.TGF-β3regulation of chondrogenesis and osteogenesis in zebrafsh is mediated through formation and survival of a subpopulation of the cranial neural crest[J].Mech Dev,2010,127(7/8):329-344.
[11]WANG Y,HE T,LIU J,et al.Synergistic effects of overexpression of BMP-2 and TGF-β3 on osteogenic differentiation of bone marrow mesenchymal stem cells[J].Mol Med Rep,2016,14(6):5514-5520.
[12]ROHAINA C M,THEN K Y,NG A M,et al.Reconstruction of limbal stem cell deficient corneal surface with induced human bone marrow mesenchymal stem cells on amniotic membrane[J].Transl Res,2014,163(3):200-210.
[2]LIU J,YU F,SUN Y,et al.Concise reviews:characteristics and potential applications of human dental tissue-derived mesenchymal stem cells[J].Stem Cells,2015,33(3):627-638.
[3]DOLE N S,MAZUR C M,ACEVEDO C,et al.Osteocyteintrinsic TGF-βsignaling regulates bone quality through perilacunar/canalicular remodeling[J].Cell Rep,2017,21(9):2585-2596.
[4]刘晓文,木合塔尔·霍加.转化生长因子-β3对兔牙髓干细胞增殖及骨向分化的影响[J].中华实用诊断与治疗杂志,2016,30(3):242-245.
[5]JOHNSEN S A,SUBRAMANIAM M,KATAGIRI T,et al.Transcriptional regulation of Smad2is required for enhancement of TGF-β/Smad signaling by TGF-βinducible early gene[J].JCell Biochem,2002,87(2):233-241.
[6]王腾,木合塔尔·霍加.外源性转化生长因子-β3联合兔牙髓干细胞对兔骨缺损处成骨细胞转化生长因子-β3表达的影响[J].中华实用诊断与治疗杂志,2017,31(6):525-529.
[7]BEEDERMAN M,LAMPLOT J D,NAN G,et al.BMPsignaling in mesenchymal stem cell differentiation and bone formation[J].J Biomed Sci Eng,2013,6(8):32-52.
[8]JUNG U W,LEE I K,PARK J Y,et al.The efficacy of BMP-2preloaded on bone substitute or hydrogel for bone regeneration at peri-implant defects in dogs[J].Clin Oral Implants Res,2015,26(12):1456-1465.
[9]TOOM A,AREND A,GUNNARSSON D,et al.Bone formation zones in heterotopic ossifications:histologic findings and increased expression of bone morphogenetic protein 2and transforming growth factors-β2 andβ3[J].Calcif Tissue Int,2007,80(4):259-267.
[10]CHEAH F S,WINKLER C,JABS E W,et al.TGF-β3regulation of chondrogenesis and osteogenesis in zebrafsh is mediated through formation and survival of a subpopulation of the cranial neural crest[J].Mech Dev,2010,127(7/8):329-344.
[11]WANG Y,HE T,LIU J,et al.Synergistic effects of overexpression of BMP-2 and TGF-β3 on osteogenic differentiation of bone marrow mesenchymal stem cells[J].Mol Med Rep,2016,14(6):5514-5520.
[12]ROHAINA C M,THEN K Y,NG A M,et al.Reconstruction of limbal stem cell deficient corneal surface with induced human bone marrow mesenchymal stem cells on amniotic membrane[J].Transl Res,2014,163(3):200-210.