持续性压力促进成骨细胞分泌VEGF的机制研究
摘要
[目的]
探讨持续性压力促进成骨细胞分泌VEGF的机制。
[方法]
1.取1～2日龄SD乳大鼠颅盖骨进行成骨细胞原代培养,后检测并鉴定成骨细胞。
2.将传代并培养至第三代的细胞分加压组和不加压组,每组再分成PD98059不预处理组和预处理组。加压组在密闭容器内采用压缩空气施以100 kPa的静压力,分别加压0.5、2.0、6.0 h。
3.各组培养基ELISA法检测VEGF浓度,提取各组细胞总蛋白,Western blot检测各组成骨细胞内磷酸化ERK1/2(phosphorylation ERK1/2, pERK1/2)的水平;提取各组细胞总mRNA,RT-PCR检测VEGF mRNA的变化。
[结果]
1.大鼠成骨细胞的培养和鉴定:原代细胞培养1d后,贴壁展开,不规则,胞核大而清楚,呈三角形或梭形,培养5-7d后细胞半融合,7-8d后,80-90%融合,“贴壁法”传代并纯化细胞后,细胞接种1h即可贴壁,生长快,3-4d即可铺满瓶底,形态与原代相同,NBT-BCIP法定性检测碱性磷酸酶发现深蓝色至蓝紫色化合物。
2.持续性压力明显增加ERK1/2的磷酸化水平;而ERK1/2总蛋白的量却无明显变化。
3.PD98059在显著抑制持续性加压所诱导的成骨细内ERK1/2磷酸化水平的同时,抑制了VEGF的mRNA的表达。
[结论]
持续性压力通过ERK1/2的活性调节成骨细胞VEGF的分泌
Objective:
To investigate the mechanism of VEGF expression in osteoblasts induced by durative pressure.
Methods:
1、osteoblast were cultured and accredited, from 1-2d SD rats to take primary cell
2、The osteoblasts were mechanically stimulated on coverslips.The phosphorylation of ERK1/2 of osteoblasts was measured by Western blot.The mRNA of VEGF was measured by RT-PCR.
Results:
1、Osteoblasts in culture and identification: Osteoblasts were isolated from the calvariae of neonatal (one day old) Sprague-Dawley rats and cultured according to published protocols.Cells be expanded and adherented, anomaly,cells’s nucleus are big and clear,and form be triangle or fusiform shape,half- coalesce after cultured 5-7d,80%-90% coalesce after cultured 7-8d, passage. NBT-BCIP method qualitation to detect AKP and acquire dark blue and amethyst compound.
2、The phosphorylation of ERK1/2 in osteoblasts treated with durative pressure significantly increased.
3、The VEGF mRNA level of osteoblasts treated with durative pressure significantly increased and this increase was inhibited after treating with PD98059.
Conclusions:
These data suggests that durative pressure induce VEGF expression in osteoblasts, which was dependent the ERK1/2 activation.
探讨持续性压力促进成骨细胞分泌VEGF的机制。
[方法]
1.取1～2日龄SD乳大鼠颅盖骨进行成骨细胞原代培养,后检测并鉴定成骨细胞。
2.将传代并培养至第三代的细胞分加压组和不加压组,每组再分成PD98059不预处理组和预处理组。加压组在密闭容器内采用压缩空气施以100 kPa的静压力,分别加压0.5、2.0、6.0 h。
3.各组培养基ELISA法检测VEGF浓度,提取各组细胞总蛋白,Western blot检测各组成骨细胞内磷酸化ERK1/2(phosphorylation ERK1/2, pERK1/2)的水平;提取各组细胞总mRNA,RT-PCR检测VEGF mRNA的变化。
[结果]
1.大鼠成骨细胞的培养和鉴定:原代细胞培养1d后,贴壁展开,不规则,胞核大而清楚,呈三角形或梭形,培养5-7d后细胞半融合,7-8d后,80-90%融合,“贴壁法”传代并纯化细胞后,细胞接种1h即可贴壁,生长快,3-4d即可铺满瓶底,形态与原代相同,NBT-BCIP法定性检测碱性磷酸酶发现深蓝色至蓝紫色化合物。
2.持续性压力明显增加ERK1/2的磷酸化水平;而ERK1/2总蛋白的量却无明显变化。
3.PD98059在显著抑制持续性加压所诱导的成骨细内ERK1/2磷酸化水平的同时,抑制了VEGF的mRNA的表达。
[结论]
持续性压力通过ERK1/2的活性调节成骨细胞VEGF的分泌
Objective:
To investigate the mechanism of VEGF expression in osteoblasts induced by durative pressure.
Methods:
1、osteoblast were cultured and accredited, from 1-2d SD rats to take primary cell
2、The osteoblasts were mechanically stimulated on coverslips.The phosphorylation of ERK1/2 of osteoblasts was measured by Western blot.The mRNA of VEGF was measured by RT-PCR.
Results:
1、Osteoblasts in culture and identification: Osteoblasts were isolated from the calvariae of neonatal (one day old) Sprague-Dawley rats and cultured according to published protocols.Cells be expanded and adherented, anomaly,cells’s nucleus are big and clear,and form be triangle or fusiform shape,half- coalesce after cultured 5-7d,80%-90% coalesce after cultured 7-8d, passage. NBT-BCIP method qualitation to detect AKP and acquire dark blue and amethyst compound.
2、The phosphorylation of ERK1/2 in osteoblasts treated with durative pressure significantly increased.
3、The VEGF mRNA level of osteoblasts treated with durative pressure significantly increased and this increase was inhibited after treating with PD98059.
Conclusions:
These data suggests that durative pressure induce VEGF expression in osteoblasts, which was dependent the ERK1/2 activation.
引文
[1] Yang SH,sharrocks AD,et al .Transcriptional regulation by the MAP kinase signaling cascades [J].Gene , 2003, 320(27):3-21.
[2] Reusch HP ,Chan G,et al. Activation of JNK/SAPK and ERK by mechanical strain in vascular smooth muscle cells depends on extracellular matrix composition[J]. Biochem Biophys Res Commun. 1997 Aug 18;237(2):239-44
[3] Gebken J ,Luders B,et al. Hypergravity stimulates collagen synthesis in human osteoblast-like cells: evidence for t he involvement of p44/42 MAP-kinases (ERK 1/2)[J]. Biochem. 1999 Oct;126(4):676-82.
[4] Wadhwa S,Godwin SL,et al Fluid flow induction of cyclo-oxygenase 2 gene expression in osteoblasts is dependent on an extracellular signal-regulated kinase signaling pathway[J]. Bone Miner Res. 2002 Feb;17(2):266-74.
[5] Laurent A ,Lantieri MD. Vascular endothelial growth factor expression in expanded tissue :A possible mechanism of angiogenesis in tissue ex2 pansion[J ] . Plast Reconstr Surg ,1998 ,101 (2) :3922397.
[6] Charlebois M, Pretterklieber M, Zysset PK.et al The role of fabric in the large strain compressive behavior of human trabecular bone[J ]Biomech Eng. 2010 Dec;132(12):121006.
[7]王前,钟世镇,龚文汇,等.鼠颅盖骨成骨细胞体外培养及ALP免疫组化法鉴别纯化[J].中华骨科杂志,1995,1(6):364-366
[8] lngber DE . Tensegrity : the architectural basis of cellular mechanotransduction[J].Annu Rev Physiol,1997,59:575—599.
[9]李可心,尚天裕,董福慧,等。间断压力对离体培养成骨细胞作用的研究[J],1999,9(5):7-9
[10] Boule GH.The biochemistry H¨d physiology-0 bone Vol 3.New York AcademicPress lnc, 1971. 1—76.
[11] Pearson G , Robinson F , Beers Gibson T , et a1 . Mitogenactivated protein(MAP)kinase pathways:regulation and physiological functions[J].Endocr Rev,2001,22(2):153—183.
[12] A. Miyagawa1, M. Chiba .et al Compressive Force Induces VEGF Production in Periodontal Tissues [J] Dent Res 88(8):752-756, 2009
[13] Mayr wohlfart U Waltenberger J Haumer H Kessler S Gunther KP,Dehio C. Puhl W,Brenner R-E Vascular endothelial growth factor stimu|ate~chemot~cdc migration of primary human osteoblasts Bone.2OO2;30(3):472_7
[14] Hayami T,Funaki H,Yaoeda K.Expression of the cartilage derived anti-angiogenic factor chondromdulin-1 decreases in the early stage of experimental osteoarthritis[J].J Rheumatol。2003,30(10):2207—2217.
[15] Ferrara N. Henzel W J. Pituitary follicular cells secrete a Novel heparin—binding g rowth factor specific for vascular endothelia cells. Biochem Biophys Res Commun,1989 16l:851-855
[1]Claes LE,Heigele CA,Ncidlinger—Wilke C,et a1.Effort of mehanical factors on the fracture healing process[J].Clin onhop, 1998,355:132—147.
[2]Claes LE,Heigele CA.Magnitudes of local stees and strain along bony surface predict the course and type of fracture healing[J].J Biomech,1999,32(3):255—266.
[3]Mcallister TN,Frangos JA.Steray and transient fluid stimelate NO release in osteobhults through distinct biochemical pathways[J].J Bone MinerRes,1999,14(6):930—936.
[4]Jorgensen NR.Short—range imercdlularcalcium signaling in bone[J].APMIS,2005,11(8):5—36.
[5]Jean Jx,Siller—Jackson AJ,Burra S.Roles of gap junction and hemichannels in bone cell functions and in signal transmission of mechnical stress[J].Front Biosci,2007,1(12):1450—1462.
[6]李继华,胡静,王志国,等.牵张成骨时整合索Bl在细胞内外张应力传递中的作用[J].口腔医学研究,2005,4(21):127—129.
[7] Basso N,Heersche JN.Characteristics of in vitro osteoblastic cell loading models[J].Bone,2002,30(2):347—351.
[8]李良,陈孟诗,邓力,等.力学刺激对大鼠成骨细胞Inte—grins grins表达和细胞增殖,合成功能的影响[J].生物医学工程杂志,2003,20(2):187—192.
[9] Cillo JE Jr,Gassner R,Koepsel RR.Growth factor and cytokine gene expression in mechanically strained human osteoblast—like cells:implications for distraction osteogenesis[J].Oral Surg Oral Med Oral Pathol Oral Radiol Ended,2000.8(2):147—154.
[10]邹淑娟,胡静.机械张力对人成骨样细胞TGF一β表达的影响[J].现代口腔医学杂志,2002,16(3):214—215.
[11]龙洁田,卫东,郑晓辉,等.张力对成骨样细胞PGE2表达的影响[J].实用口腔医学杂志,2005,3(2):160—162.
[12]Sanuki R,Mitsui N,Suzuki N,et a1.Effect ofcompressive force on the production ofprostaglandin E(2)and its receptors in osteblastie Saos-2 cells[J].Connect Tissue Res,2007,48(5):246—253.
[13]鲜成玉,王远亮,张兵兵,等.机械拉伸对成骨细胞增殖及IGF—I mRNA表达的影响[J].生物医学工程学杂志,2007,24(2):312—315..
[14]Zhang K,Barragan Adjemian c,Ye L,ct a1.E11/g P38 selective expression in osteecytes:regulation by mechanical strain and role in dendrite elongation[J].Mol Cell Bid。2006,26(12):4539—4552.
[15] Kenwright J,Gardner T.Mechanical influence on tibial fracture heali. [J].Clin Orthop,1998,335:179—190.
[16]Chao EYS,lnoue N,Elias JJ,ct a1.Enhancement of fracture healing by mechanical and surgical intervention[J].Clin Orthop,1998,335:163—178.
[17]Claes LE,Heigele CA,Neidlinger—Wilke C,et a1.Effects of mechanieal factors on the fracture healing process[J].C]in Orthop,1998,355:132—147.
[18]Claes LE,Heigele CA.Magnitudes of local stress and strain along bony surfaces predict the course and type of fracture healing[J].J Biomech,1999,32(3):255—266.
[19]McALllster TN,Franges JA.Steady and transient fluid shear stress stinulate NO release in osteoblasts through distinct biochemical pathways[J].J Bone Miner Res,1999,14(6):930-936.
[20]Gardner TN,Stoll T,Marks L.The influence of mechanical stimulus on the pattern of tissue differentiation in a long bone fracture—an FEM study[J].J Biomech,2000,33(4):415—425.
[21]Loboa EG,Beaupre GS,Carter DR.Mechanobiology of initial pseudarthrosis formation with oblique fractures[J].J Orthop Res,2001,19(6):1067—1072.
[22]Augat P,Burger J,Sehorlemmer S,et a1.Shear movement at the fracture sitedelays healing in a diaphyseal fracture model[J].J Orthop Res,2003,2l(6):1011—1017
[23]喻鑫罡,张先龙,曾炳芳.骨折段低频可控微动影响骨痂矿化与力学特征的实验研究[J].上海交通大学学报(医学版),2008,28(12):1491—1495.
[24]Page—McCaw A,Ewald AJ,Werb z.Matrix metalloproteinases and the regulation of tissue remodeling[J].Nat Rew Mol Cell Bi—ol,2007,8(3):221—233.
[25]Here P,Zimmermann G,Weiss S.Matrix metalloproteinases and failed fracture healing[J].Bone,2005,37(6):791-798.
[2] Reusch HP ,Chan G,et al. Activation of JNK/SAPK and ERK by mechanical strain in vascular smooth muscle cells depends on extracellular matrix composition[J]. Biochem Biophys Res Commun. 1997 Aug 18;237(2):239-44
[3] Gebken J ,Luders B,et al. Hypergravity stimulates collagen synthesis in human osteoblast-like cells: evidence for t he involvement of p44/42 MAP-kinases (ERK 1/2)[J]. Biochem. 1999 Oct;126(4):676-82.
[4] Wadhwa S,Godwin SL,et al Fluid flow induction of cyclo-oxygenase 2 gene expression in osteoblasts is dependent on an extracellular signal-regulated kinase signaling pathway[J]. Bone Miner Res. 2002 Feb;17(2):266-74.
[5] Laurent A ,Lantieri MD. Vascular endothelial growth factor expression in expanded tissue :A possible mechanism of angiogenesis in tissue ex2 pansion[J ] . Plast Reconstr Surg ,1998 ,101 (2) :3922397.
[6] Charlebois M, Pretterklieber M, Zysset PK.et al The role of fabric in the large strain compressive behavior of human trabecular bone[J ]Biomech Eng. 2010 Dec;132(12):121006.
[7]王前,钟世镇,龚文汇,等.鼠颅盖骨成骨细胞体外培养及ALP免疫组化法鉴别纯化[J].中华骨科杂志,1995,1(6):364-366
[8] lngber DE . Tensegrity : the architectural basis of cellular mechanotransduction[J].Annu Rev Physiol,1997,59:575—599.
[9]李可心,尚天裕,董福慧,等。间断压力对离体培养成骨细胞作用的研究[J],1999,9(5):7-9
[10] Boule GH.The biochemistry H¨d physiology-0 bone Vol 3.New York AcademicPress lnc, 1971. 1—76.
[11] Pearson G , Robinson F , Beers Gibson T , et a1 . Mitogenactivated protein(MAP)kinase pathways:regulation and physiological functions[J].Endocr Rev,2001,22(2):153—183.
[12] A. Miyagawa1, M. Chiba .et al Compressive Force Induces VEGF Production in Periodontal Tissues [J] Dent Res 88(8):752-756, 2009
[13] Mayr wohlfart U Waltenberger J Haumer H Kessler S Gunther KP,Dehio C. Puhl W,Brenner R-E Vascular endothelial growth factor stimu|ate~chemot~cdc migration of primary human osteoblasts Bone.2OO2;30(3):472_7
[14] Hayami T,Funaki H,Yaoeda K.Expression of the cartilage derived anti-angiogenic factor chondromdulin-1 decreases in the early stage of experimental osteoarthritis[J].J Rheumatol。2003,30(10):2207—2217.
[15] Ferrara N. Henzel W J. Pituitary follicular cells secrete a Novel heparin—binding g rowth factor specific for vascular endothelia cells. Biochem Biophys Res Commun,1989 16l:851-855
[1]Claes LE,Heigele CA,Ncidlinger—Wilke C,et a1.Effort of mehanical factors on the fracture healing process[J].Clin onhop, 1998,355:132—147.
[2]Claes LE,Heigele CA.Magnitudes of local stees and strain along bony surface predict the course and type of fracture healing[J].J Biomech,1999,32(3):255—266.
[3]Mcallister TN,Frangos JA.Steray and transient fluid stimelate NO release in osteobhults through distinct biochemical pathways[J].J Bone MinerRes,1999,14(6):930—936.
[4]Jorgensen NR.Short—range imercdlularcalcium signaling in bone[J].APMIS,2005,11(8):5—36.
[5]Jean Jx,Siller—Jackson AJ,Burra S.Roles of gap junction and hemichannels in bone cell functions and in signal transmission of mechnical stress[J].Front Biosci,2007,1(12):1450—1462.
[6]李继华,胡静,王志国,等.牵张成骨时整合索Bl在细胞内外张应力传递中的作用[J].口腔医学研究,2005,4(21):127—129.
[7] Basso N,Heersche JN.Characteristics of in vitro osteoblastic cell loading models[J].Bone,2002,30(2):347—351.
[8]李良,陈孟诗,邓力,等.力学刺激对大鼠成骨细胞Inte—grins grins表达和细胞增殖,合成功能的影响[J].生物医学工程杂志,2003,20(2):187—192.
[9] Cillo JE Jr,Gassner R,Koepsel RR.Growth factor and cytokine gene expression in mechanically strained human osteoblast—like cells:implications for distraction osteogenesis[J].Oral Surg Oral Med Oral Pathol Oral Radiol Ended,2000.8(2):147—154.
[10]邹淑娟,胡静.机械张力对人成骨样细胞TGF一β表达的影响[J].现代口腔医学杂志,2002,16(3):214—215.
[11]龙洁田,卫东,郑晓辉,等.张力对成骨样细胞PGE2表达的影响[J].实用口腔医学杂志,2005,3(2):160—162.
[12]Sanuki R,Mitsui N,Suzuki N,et a1.Effect ofcompressive force on the production ofprostaglandin E(2)and its receptors in osteblastie Saos-2 cells[J].Connect Tissue Res,2007,48(5):246—253.
[13]鲜成玉,王远亮,张兵兵,等.机械拉伸对成骨细胞增殖及IGF—I mRNA表达的影响[J].生物医学工程学杂志,2007,24(2):312—315..
[14]Zhang K,Barragan Adjemian c,Ye L,ct a1.E11/g P38 selective expression in osteecytes:regulation by mechanical strain and role in dendrite elongation[J].Mol Cell Bid。2006,26(12):4539—4552.
[15] Kenwright J,Gardner T.Mechanical influence on tibial fracture heali. [J].Clin Orthop,1998,335:179—190.
[16]Chao EYS,lnoue N,Elias JJ,ct a1.Enhancement of fracture healing by mechanical and surgical intervention[J].Clin Orthop,1998,335:163—178.
[17]Claes LE,Heigele CA,Neidlinger—Wilke C,et a1.Effects of mechanieal factors on the fracture healing process[J].C]in Orthop,1998,355:132—147.
[18]Claes LE,Heigele CA.Magnitudes of local stress and strain along bony surfaces predict the course and type of fracture healing[J].J Biomech,1999,32(3):255—266.
[19]McALllster TN,Franges JA.Steady and transient fluid shear stress stinulate NO release in osteoblasts through distinct biochemical pathways[J].J Bone Miner Res,1999,14(6):930-936.
[20]Gardner TN,Stoll T,Marks L.The influence of mechanical stimulus on the pattern of tissue differentiation in a long bone fracture—an FEM study[J].J Biomech,2000,33(4):415—425.
[21]Loboa EG,Beaupre GS,Carter DR.Mechanobiology of initial pseudarthrosis formation with oblique fractures[J].J Orthop Res,2001,19(6):1067—1072.
[22]Augat P,Burger J,Sehorlemmer S,et a1.Shear movement at the fracture sitedelays healing in a diaphyseal fracture model[J].J Orthop Res,2003,2l(6):1011—1017
[23]喻鑫罡,张先龙,曾炳芳.骨折段低频可控微动影响骨痂矿化与力学特征的实验研究[J].上海交通大学学报(医学版),2008,28(12):1491—1495.
[24]Page—McCaw A,Ewald AJ,Werb z.Matrix metalloproteinases and the regulation of tissue remodeling[J].Nat Rew Mol Cell Bi—ol,2007,8(3):221—233.
[25]Here P,Zimmermann G,Weiss S.Matrix metalloproteinases and failed fracture healing[J].Bone,2005,37(6):791-798.