应变对间充质干细胞向成骨细胞分化的力学响应机制研究
|
摘要
目的：利用力学加载装置对小鼠骨髓间充质干细胞系(D1细胞)加载不同拉伸应变，探讨力学因素对干细胞向成骨细胞分化的影响及力学信号转导机制。
方法：在成骨细胞诱导体系条件下，D1细胞施加不同的拉伸应变后，采用免疫组化方法、RT-PCR技术、Flou-3-AM Ca~(2+)染色方法及激光共聚焦技术。分析与成骨细胞分化有关的OSX(Osterx)、骨钙蛋白(Osteocalcin OCN)基因、碱性磷酸酶(alkaline phosphatase，ALP)、Ⅰ型胶原(type Ⅰ collagen，COLl)、OCN蛋白的表达；对细胞内ca~(2+)水平及微丝结构进行考察；利用SB203580(p38MAPK特异抑制剂)、PD98059(MEK-1／2MAPK特异抑制剂)、LY294002(PI_3KS特异抑制剂)、细胞松弛素B(微丝结构阻断剂)、EGTA(Ca~(2+)螯合剂)信号阻断剂探讨力学信号传导的信号途径。
结果：间充质干细胞向成骨细胞分化过程中，拉伸应变能促进细胞ALP、Ⅰ型胶原、OCN蛋白的表达，激活与成骨细胞分化起关键作用的OSX、OCN基因；细胞受到应变作用时，骨架结构微丝发生断裂和重排，同时细胞生长方向发生相应改变；拉伸应变能促进干细胞内Ca~(2+)水平的增加和钙火花的释放；力学信号的传导与磷酸化的磷脂酰肌醇(Phosphatidylinositol-3-kinases，PI_3Ks)信号通路、细胞骨架结构微丝、细胞内Ca~(2+)水平有密切关系。
结论：不同拉伸应变能促进间充质干细胞向成骨细胞分化；力学信号通过Ca~(2+)信号、细胞微丝结构以及PI_3Ks信号途径引起细胞的应答反应和生物学效应。
Objective To study the effect of cyclic substrate deformation on the mechanical signaling pathway of mouse Mesenchymal Stem Cells(MSCs) line(Dl). Methods Cyclic strain(3% 0.5Hz) was applied to mouse MSCs in osteogenic media. The ways of immunocytochemistry, RT-PCR were used to analyse the gene expression of OSX(Osterx),osteocalcin (OCN) ,and the protein of alkaline phosphatase (ALP)、 type I collagen (COL1) and OCN; the structure of microfilament and Ca~(2+) levels were detected by confocal laser scanning microscope (CLSM); The signaling inhabitors,such as SB203580 ( p38MAPK specific inhabitor ) 、 PD98059
(MEK-1/2MAPK specific inhabitor)、 LY294002 (PI_3Ks specific inhabitor )、 Cytochalasin B (microfilament specific inhabitor)、 EGTA (Ca~(2+)specific inhabitor) , were used to investigate mechanical signaling pathway.
Results The cyclic substrate deformation could stimulate increasing expression of ALP、 type I collagen、 OCN and activate the gene expression of OSX and OCN over unstrained cells.Mechanical strain changed the structure of microfilament and cell reorientation, and promoted obviously Ca~(2+) levels. Ca~(2+) sparks releasing were observed with fluo-3-AM by strain-induced. Strain-induced the expression of mRNA of OSX,OCN was largely mediated by Ca~(2+) signaling and Phosphatidylinositol-3-kinases
(PI_3Ks) pathway.
Conclusions Strain can enhance differentiation of mesenchymalstem cells to osteogen . Microfilament, Ca~(2+) signaling and PI_3Ks pathway play an important role on strain-induced mesenchymal stem cells differentiation.
方法：在成骨细胞诱导体系条件下，D1细胞施加不同的拉伸应变后，采用免疫组化方法、RT-PCR技术、Flou-3-AM Ca~(2+)染色方法及激光共聚焦技术。分析与成骨细胞分化有关的OSX(Osterx)、骨钙蛋白(Osteocalcin OCN)基因、碱性磷酸酶(alkaline phosphatase，ALP)、Ⅰ型胶原(type Ⅰ collagen，COLl)、OCN蛋白的表达；对细胞内ca~(2+)水平及微丝结构进行考察；利用SB203580(p38MAPK特异抑制剂)、PD98059(MEK-1／2MAPK特异抑制剂)、LY294002(PI_3KS特异抑制剂)、细胞松弛素B(微丝结构阻断剂)、EGTA(Ca~(2+)螯合剂)信号阻断剂探讨力学信号传导的信号途径。
结果：间充质干细胞向成骨细胞分化过程中，拉伸应变能促进细胞ALP、Ⅰ型胶原、OCN蛋白的表达，激活与成骨细胞分化起关键作用的OSX、OCN基因；细胞受到应变作用时，骨架结构微丝发生断裂和重排，同时细胞生长方向发生相应改变；拉伸应变能促进干细胞内Ca~(2+)水平的增加和钙火花的释放；力学信号的传导与磷酸化的磷脂酰肌醇(Phosphatidylinositol-3-kinases，PI_3Ks)信号通路、细胞骨架结构微丝、细胞内Ca~(2+)水平有密切关系。
结论：不同拉伸应变能促进间充质干细胞向成骨细胞分化；力学信号通过Ca~(2+)信号、细胞微丝结构以及PI_3Ks信号途径引起细胞的应答反应和生物学效应。
Objective To study the effect of cyclic substrate deformation on the mechanical signaling pathway of mouse Mesenchymal Stem Cells(MSCs) line(Dl). Methods Cyclic strain(3% 0.5Hz) was applied to mouse MSCs in osteogenic media. The ways of immunocytochemistry, RT-PCR were used to analyse the gene expression of OSX(Osterx),osteocalcin (OCN) ,and the protein of alkaline phosphatase (ALP)、 type I collagen (COL1) and OCN; the structure of microfilament and Ca~(2+) levels were detected by confocal laser scanning microscope (CLSM); The signaling inhabitors,such as SB203580 ( p38MAPK specific inhabitor ) 、 PD98059
(MEK-1/2MAPK specific inhabitor)、 LY294002 (PI_3Ks specific inhabitor )、 Cytochalasin B (microfilament specific inhabitor)、 EGTA (Ca~(2+)specific inhabitor) , were used to investigate mechanical signaling pathway.
Results The cyclic substrate deformation could stimulate increasing expression of ALP、 type I collagen、 OCN and activate the gene expression of OSX and OCN over unstrained cells.Mechanical strain changed the structure of microfilament and cell reorientation, and promoted obviously Ca~(2+) levels. Ca~(2+) sparks releasing were observed with fluo-3-AM by strain-induced. Strain-induced the expression of mRNA of OSX,OCN was largely mediated by Ca~(2+) signaling and Phosphatidylinositol-3-kinases
(PI_3Ks) pathway.
Conclusions Strain can enhance differentiation of mesenchymalstem cells to osteogen . Microfilament, Ca~(2+) signaling and PI_3Ks pathway play an important role on strain-induced mesenchymal stem cells differentiation.
引文
[1] Pittenger, M F, Mackay, A M, Beck,S C, et al. Multilineage potential of adult human mesenchymal stem cells[J]. Science, 1999,284:143-147.
[2] Oreffo ROC,Virdi A S,Triffit J T. Modulation of osteogenesis and adipogenesis by human serum in human bone marrow cultures[J]. Eur J Cell Biol, 1997,74:251-261.
[3] Bane A J, Tsuzald M,yamamoto J ,et al. Mechanoreception at the cellular level:the detection, interpretation, and diversity of responses to mechanical signals[J].Biochemistry and Cell Biology, 1995,73:1-16.
[4] Inger D E.Structure of life[J].Sci,An, 1998,278(1):6-15.
[5] 张毅弈，陶祖莱．载荷诱导骨生长的力学细胞生物学机制[J]．力学进展，2000，30(3)，433-445．
[6] Carmeliet G, and Bouillon R. The effect of microgravity on morphology and gene expression of osteoblasts in vitro [J].FASEB J, 1999,13:S129-S134.
[7] Kaspar D,Seidl W, Neidlinger W C ,et al. Dynamic cell stretching increases human osteoblast proliferation and CICP synthesis but decreases osteocalcin synthesis and alkaline phosphatase activity[J]. J Biomech,2000,33:45-51.
[8] Altman G H, Horan R L, Martin I, et al.. Cell differentiation by mechanical stress. FASEB.J, 2001,28:1096.
[9] Simmons C A, Matlis S, Thomton A J. Cyclic strain enhances matrix minerlization by adult human mesenchymal stem cell cells via the extracellular signal-regulated kinase(ERK1/2) signaling pathway. J. Binmech, 2001,36:1087-1096.
[10] Lian J B, Javed A, Zaidi S K, Lengner C, et al. Regulatory controls for osteoblast growth and differentiation: role of Runx/Cbfa/AML factors[J]. Crit Rev Eukaryot Gene Expr.,2004; 14(1-2):1-41.
[11] Ziros P G, Gil A P, Georgakopoulos T, et al. The bone-specific transcriptional regulator Cbfal is a target of mechanical signals in osteoblastic cells[J]. J Biol Chem, 2002,277(26):23934-41.
[12] Cui W, Bryant MR, Sweet PM,McDonnell Pj. Changes in gene\expression in response to mechanical strain in human scleral fibroblasts[J]. Exp Eye Res,2004,78(2):275-84.
[13] Berry CC,Shelton JC,Bader DL,et al. Influence of external uniaxial cyclic strain on oriented fibroblast-seeded collagen gels[J]. Tissue Eng,2003,9(4) :613-622.
[14] 卢晓，王红兵，黄垲平，等．大鼠血管平滑肌细胞形态和DNA合成对基底膜周期性应变的响应[J]．中国生物医学工程学报，2002，21(1)：16-20．[15] 秦廷武，杨志明，解慧琪，等．动态应变下肌腱细胞三维培养的初步研究[J]．华西医科大学学报，2002,33(1)：1-4．
[16] 唐丽灵，王远亮，谷俐，等．不同应变拉伸水平对成骨细胞生理功能的影响[J]．重庆大学学报，2003，3(2)：67-70．
[17] 唐丽灵，王远亮，谷俐，等．成骨细胞对梯度拉伸应变的响应[J]．生物物理学报，2003，19(1)：89-91．
[18] 张西正，康少华，匡震邦，等．单向循环拉伸应变作用下成骨细胞动力学响应的实验研究[J]．中华创伤杂志，2001，17(4)：219-221．
[19] Neelam Jw, Stephen E, Haynesworth ,et al. osteogenic differentiation of purified culture-expanded human mesenchymal stem cells in vitro[J].Journalof cellular biochemistry, 1997,64:295-312.
[20] Li X,Jin L,Wang GJ ,et al. Steroid effects on osteogenesis through mesenchymal cell gene expression[J]. Osteoporos Int,2005 16(1): 101-108.
[21] Ryan MP, William R,Huckle, et al. Effect of dexamethason withdrawal onosteoblastic differentiation of bone marrow stromal cells[J]. Journal of cellular biochemistry,2003,90:13-22.
[22] Renny T, Franceschi and Guozhi Xiao. Regulation of the osteoblast- specisic transcription factor ,Runx2:responsiveness to mulitiple signal transduction pathways[J].Journal of cellular biochemistry,2003,88:446-454.
[23] Choi K Y, Kim H J, Lee M H, et al. Runx2 regulates FGF2-induced Bmp2 expression during cranial bone development[J]. Dev Dyn. 2005,233(1): 115-21.
[24] Celil A B, Hollinger J O, Campbell PG. Osx transcriptional regulation is mediated by additional pathways to BMP2/Smad signaling[J]. J Cell Biochem, 2005,95(3):518-28.
[25] Igarashi M, Kamiya N, Hasegawa M et al. Inductive effects of dexamethasone on the gene expression of Cbfal, Osterix and bone matrix proteins during differentiation of cultured primary rat osteoblasts[J]. J Mol Histol,2004,35 (1):3-10.
[26] Woodbury D, Schwarz E J, Prockop D J, et al. Adult rat and human bone marrow stromal cells differentiate into neurons[J]. Journal of Neuroscience Research,2000,61:364-370.
[27] Xudong LI,Quanjun Cui,Chinghai Kao, et al. Lavastatin inhibits adipogenic and stimulates osteogenic differentiation by suppressing PPAR_γ, and increasing Cbfa1/Runx2 expression in bone marrow mesenchymal cell cultures[J]. Bone, 2003,33:652-659.
[28] Miura M, Chen X D, Allen M R, et al. A crucial role of caspase-3 in osteogenic differentiation of bone marrow stromal stem cells[J]. J Clin Invest, 2004,114(12):1704-13.
[29] Bielby R C, Boccaccini A R, Polak JM,et al. In vitro differentiation and in vivo mineralization of osteogenic cells derived from human embryonic stem cells[J]. Tissue Eng, 2004, 10(9-10): 1518-25.[30] Reddi A H. Extracellular matrix and development[J].Extracellular Matrix Biochemistry ,1984,247-291.
[31] Reddi AH. Morphogenesis and tissue engineering of bone and cartilage: inductive signal,stem cell, and biomimetic biomaterials[J]. Tissue Engineering,2000,6(4):351-359.
[32] Ma S, Chen G, and Reddi A H. Collaboration between collagenous matrix and osteogenin is required for bone induction[J]. Ann.N.Y.Acad.Sci,1990,580:524-525.
[33] Ryan MP, William R, Huckle, et ah Effect of dexamethason withdrawal onosteoblastic differentiation of bone marrow stromal cells[J]. Journal of cellular biochemistry,2003,90:13-22.
[34] Otto F,Lubbert M,and Stock M. Upstream and downstream targets of Runx proteins[J]. Cell Biochem, 2003,89:9-18.
[35] Dong J, Uemura T, Kikuchi M, Tanaka J, et al. Long-term durability of porous hydroxyapatite with low-pressure system to support osteogenesis of mesenchymal stem cells[J]. Biomed Mater Eng, 2002;12(2):203-9.
[36] Nishimura H, Nishimura M, Oda R, Yamanaka K, et ah Lectins induce resistance to proteases and/or mechanical stimulus in all examined cells--including bone marrow mesenchymal stem cells--on various scaffolds[J]. Exp Cell Res, 2004 Apr 15;295(1):119-27.
[37] Laboureau J, Dubertret L, Lebreton D C, et al. ERK activation by mechanical strain is regulated by the small G proteins rac-1 and rhoA[J]. Exp.Derma, 2004.13:70-77.
[38] Ziros P G, Gil A P, Georgakopoulos T, et al. The bone-specific transcriptional regulator Cbfa1 is a target of mechanical signals in osteoblastic cells[J]. J Biol Chem, 2002,277(26):23934-41.
[39] Costessi A, Pines A, D'Andrea P, et al. Extracellular nucleotides activate Runx2 in the osteoblast-like HOBIT cell line: a possible molecular link between mechanical stress and osteoblasts' response[J]. Bone,2005 ,36(3):418-32.
[40] Altaian G H, Horan R L, Martin I, et al. Cell differentiation by mechanical stress. FASEB.J,2001,28:1096.
[41] Raab-Cullen D M, Thiede M A, Peterson D N, et al. Mechanical loading stimulates rapid changes in periosteal gen expression[J]. Calcif Tiss Int,1994,55:473-478.
[42] Inaoka T,Lean J M, Bessho T, et al. Sequential analysis of gene expression after an osteogenic stimulus:c-fos expression is induced in osteocytes[J]. Biochem Biophys Res Commun,1995,217:264-270.
[43] Wong M, Siegrist M, Goodwin K. Cyclic tensile strain and cyclic hydrostatic pressure differentially regulate expression of hypertrophic markers in primary chondrocytes[J]. Bone, 2003 ,33(4):685-93.[44] Wang M D. Manipulation of single molecules in biology[J]. Curr Opin Biotechnol,1999,10(1):81-6.
[45] Ahdjoudj S, Lasmoles F, Holy X, et al. Transforming growth factor beta2 inhibits adipocyte differentiation induced by skeletal unloading in rat bone marrow stroma[J]. J Bone Miner Res,2002, 17(4):668-77.
[46] Shigeo M T, Jiliang Li, Randall L, et al. Effects of broad frequency vibration on cultured osteoblasts[J].Journal of Biomechanics,2003,36:73-80.
[47] Karmo T, Takahashi T, Ariyoshi W, et al. Tensile mechanical strain up-regulates Runx2 and osteogenic factor expression in human periosteal cells: implications for distraction osteogenesis[J]. J Oral Maxillofac Surg. 2005,63(4):499-504.
[48] Ignatius A, Blessing H, Liedert A, et al. Effects of mechanical strain on human osteoblastic precursor cells in type I collagen matrices[J]. Orthopade. 2004,33(12): 1386-93.
[49] Koike M, Shimokawa H, Kanno Z et al. Effects of mechanical strain on proliferation and differentiation of bone marrow stromal cell line ST2[J]. J Bone Miner Metab, 2005;23(3):219-25.
[50] 翟中和，王喜忠，丁明孝，主编．细胞生物学[M]．北京：高等教育出版社，2000，319-343．
[51] Wang N, Ingber D E. Control of cytoskeletal mechanics by extracellular matrix, cell shape, and mechanical tension[J]. Biophysical Journal, 1994,66:2181-2189.
[52] Jennifer S P, Julia S F, Catherine Cheng et al. Differential effects of equiaxial and uniaxial strain on mesenchymal stem cells[J]. Biotechnology and Bioengineering, 2004,88 (3):360-368.
[53] Wang N,Vujak-Novakovik G.Mechanotransduction across the cell surface and through the cytoskeleton[J]. Science, 1993, 260:1124-17.
[54] Meazzini M C,Toma C D,Schaffer I L,et al.Ostoeblast cytoskeletal modulation in response to mechanical strain in vitro[J]. J Orthop Res, 1998, 16(2)170-80.
[55] 杨美祥，林珠，丁寅等．不同应力对人牙周膜成纤维细胞细胞骨架影响的体外研究[J]．中国临床康复，20013，7(23)：3162-63．
[56] Banneyx G, Baugh L, Vogel V. Fibronectin extension and unfolding with in cell matrix fibrils controlled by cytoskeletal tension[J]. Proc Natl Acad Sci USA, 2002, 99(8):5139-43.
[57] Takemasa T, Sugimoto K,Yamashita K. Amplitude-dependent stress fiber reorientation in early response to cyclic strain[J]. Experimental Cell Research, 1997.77:91-99.
[58] 霍霞，吕建勋，杨仁东等．早期人羊膜共聚焦激光扫描光学切片及F-肌动蛋白的研究[J]．解剖学报，2002，33(6)：283-87．
[59] Dartsch P C, Betz E. Response of cultured endothelial cells to mechanical stimulation[J]. Basic Research in Cardiology, 1989, 84:268-281.[60] James H C, Pascal G C, Jeremiah Wille, et al. Specificity of endothelial cell reorientation in response to cyclic mechanical stretching[J].Journal of Biomechanics,2001,34:1563-1572.
[61] 刘景生 主编．细胞信息与调控{M}．北京医科大学、中国协和医科大学联合出版社，北京．187-200
[62] Duncan R L, Hatter L V, Levin D W, et al. Regulation of strech-activated cation channel activity via the cytoskeleton and similar to hormonal regulation. Mol Biol Cell, 1992,3:38a.
[63] Cheng H,Lederer W J,Cannel M B. Calcium sparks:elementary events underlying excitation-contraction coupling in heart muscle[J].science,1993,262:740-744.
[64] Isaeva E V, Shkryl VM,Shirokova N. Mitochondrial redox state and Ca~(2+) sparks in permeabilized mammalian, skeletal muscle[J].J Physiol,2005,565:855-872.
[65] Kamishima T, Quayle J M. Ca2+-induced Ca~(2+) release in cardiac and smoothmucclecells.BiochemicalSocietyTransactions,2003,31:943-946
[66] Guharay F, Sashs F. Stretch-activated single ion channel current in tissue cultured embryonic chick skeletal muscle[J]. 1984,352:685-701.
[67] Petkov G V, Nelson M T. Differential regulation of Ca2+-activated K+ channels by beta-adrenoceptors in guinea pig urinary bladder smooth muscle[J].Am J Physiol Cell Physiol,2005,288(6):C1255-63.
[68] Walker L M,Publicover S J,Preston M R, et al. Calcium-channel activation and matrix protein upregulation in bone cells in response to mechanical strain[J].Joumal of Cellular Biochemistry,2000,79:648-661.
[69] Xia S L, Ferrier J. Calcium signal induced by mechanical perturbation of osteoclasts[J].J Cell Physiol, 1995,163:493-501.
[70] Zaman G;Suswillo RFL,Cheng M Z, et al. Early responses to dynamic strain changes and prostaglandins in bone-derived cells in culture[J]. J Bone Miner Res ,1997,12:132-136.
[71] Li X, Zima A V, Sheikh F, et al. Endothelin-1-induced arrhythrnogenic Ca~(2+) signaling is abolished in atrial myocytes of inositol-1,4,5- trisphosphateTP3)-receptor type 2-deficient mice[J].Circ Res, 2005, 96(12): 1:274-81.
[72] Chandler W.K, Hollingworth S and S.M. Baylor. Simulation of Calcium Sparks in Cut Skeletal Muscle Fibers of the Frog[J]. Journal of General Physiology, 2003,121 (4):311-324.
[73] 张钦，程和平．心肌细胞钙火花[J]．中国药理学通报．2005，21(1)：23-26．
[74] Zhou J, Brum G, Gonzalez A, et al. Ca2+ sparks and embers of mammalian muscle. Properties of the sources[J] .J Gen Physiol, 2003,122(1):95-114
[75] Inone M,and Bridge J H. Ca2+ sparks in rabbit ventricular myocytes evoked by action potentials: involvement of clusters of L-type Ca2+ channels[J]. Circ Res,2003,92(5):532-8.[76] Jessop H L,and Rawlinson S C, Pitsillides AA et al. Mechanical strain and fluid movement both activate extracellular regulated kinase(ERK) in osteoblast like cells but via different signaling pathways. Bone, 2002,31:186-194.
[77] Long.P, Liu.F, Piesco.N P, et al. 2002.Signaling by mechanical strain involves transcriptional regulation of proinflammatory genes in human periodontal ligament cellsin vitro. Bone,30:547-552.
[78] Laboureau J,Dubertret L, Lebreton D C, et al. ERK activation by mechanical strain is regulated by the small G proteins rac-1 and rhoA. Exp.Derma, 2004,13:70-77.
[79] Kanno T, Takahashi T, Ariyoshi W, et al. Tensile mechanical strain up-regulates Runx2 and osteogenic factor expression in human periosteal cells: implications for distraction osteogenesis[J]. J Oral Maxillofac Surg, 2005 ,63(4):499-504.
[80] Liang F, and Gardner D G Mechanical strain activates BNP gene transcription through a p38/NF-kappa-B-dependent mechanism[J]J.Clin. Invest, 1999.104,1603-1612.
[81] Carey M B,and Matsumoto S GCalcium transient activity in culured murine neural crest cells is regulated at IP3 receptor[J] .Brain Research,2000,862:201-210.
[82] Carpio L,Gladu J,Goltzman D et al. Induction of osteoblast differentiation indexes by PTHrP in MG-63 cells involves multiple signaling pathways[J]. Am J Physiol Endocrinol Metab, 2001,281:E489-99.
[83] Jessop H L, Rawlinson S C, Pitsillides AA,et al. Mechanical strain and fluid movement both activate extracellular regulated kinase(ERK) in osteoblast like cells but via different signaling pathways[J]. Bone, 2002,31:186-194.
[84] Berridge MJ. The biology and medicin of calcium signaling. Mol Cellular Endocri,1994,98:119-1
[2] Oreffo ROC,Virdi A S,Triffit J T. Modulation of osteogenesis and adipogenesis by human serum in human bone marrow cultures[J]. Eur J Cell Biol, 1997,74:251-261.
[3] Bane A J, Tsuzald M,yamamoto J ,et al. Mechanoreception at the cellular level:the detection, interpretation, and diversity of responses to mechanical signals[J].Biochemistry and Cell Biology, 1995,73:1-16.
[4] Inger D E.Structure of life[J].Sci,An, 1998,278(1):6-15.
[5] 张毅弈，陶祖莱．载荷诱导骨生长的力学细胞生物学机制[J]．力学进展，2000，30(3)，433-445．
[6] Carmeliet G, and Bouillon R. The effect of microgravity on morphology and gene expression of osteoblasts in vitro [J].FASEB J, 1999,13:S129-S134.
[7] Kaspar D,Seidl W, Neidlinger W C ,et al. Dynamic cell stretching increases human osteoblast proliferation and CICP synthesis but decreases osteocalcin synthesis and alkaline phosphatase activity[J]. J Biomech,2000,33:45-51.
[8] Altman G H, Horan R L, Martin I, et al.. Cell differentiation by mechanical stress. FASEB.J, 2001,28:1096.
[9] Simmons C A, Matlis S, Thomton A J. Cyclic strain enhances matrix minerlization by adult human mesenchymal stem cell cells via the extracellular signal-regulated kinase(ERK1/2) signaling pathway. J. Binmech, 2001,36:1087-1096.
[10] Lian J B, Javed A, Zaidi S K, Lengner C, et al. Regulatory controls for osteoblast growth and differentiation: role of Runx/Cbfa/AML factors[J]. Crit Rev Eukaryot Gene Expr.,2004; 14(1-2):1-41.
[11] Ziros P G, Gil A P, Georgakopoulos T, et al. The bone-specific transcriptional regulator Cbfal is a target of mechanical signals in osteoblastic cells[J]. J Biol Chem, 2002,277(26):23934-41.
[12] Cui W, Bryant MR, Sweet PM,McDonnell Pj. Changes in gene\expression in response to mechanical strain in human scleral fibroblasts[J]. Exp Eye Res,2004,78(2):275-84.
[13] Berry CC,Shelton JC,Bader DL,et al. Influence of external uniaxial cyclic strain on oriented fibroblast-seeded collagen gels[J]. Tissue Eng,2003,9(4) :613-622.
[14] 卢晓，王红兵，黄垲平，等．大鼠血管平滑肌细胞形态和DNA合成对基底膜周期性应变的响应[J]．中国生物医学工程学报，2002，21(1)：16-20．[15] 秦廷武，杨志明，解慧琪，等．动态应变下肌腱细胞三维培养的初步研究[J]．华西医科大学学报，2002,33(1)：1-4．
[16] 唐丽灵，王远亮，谷俐，等．不同应变拉伸水平对成骨细胞生理功能的影响[J]．重庆大学学报，2003，3(2)：67-70．
[17] 唐丽灵，王远亮，谷俐，等．成骨细胞对梯度拉伸应变的响应[J]．生物物理学报，2003，19(1)：89-91．
[18] 张西正，康少华，匡震邦，等．单向循环拉伸应变作用下成骨细胞动力学响应的实验研究[J]．中华创伤杂志，2001，17(4)：219-221．
[19] Neelam Jw, Stephen E, Haynesworth ,et al. osteogenic differentiation of purified culture-expanded human mesenchymal stem cells in vitro[J].Journalof cellular biochemistry, 1997,64:295-312.
[20] Li X,Jin L,Wang GJ ,et al. Steroid effects on osteogenesis through mesenchymal cell gene expression[J]. Osteoporos Int,2005 16(1): 101-108.
[21] Ryan MP, William R,Huckle, et al. Effect of dexamethason withdrawal onosteoblastic differentiation of bone marrow stromal cells[J]. Journal of cellular biochemistry,2003,90:13-22.
[22] Renny T, Franceschi and Guozhi Xiao. Regulation of the osteoblast- specisic transcription factor ,Runx2:responsiveness to mulitiple signal transduction pathways[J].Journal of cellular biochemistry,2003,88:446-454.
[23] Choi K Y, Kim H J, Lee M H, et al. Runx2 regulates FGF2-induced Bmp2 expression during cranial bone development[J]. Dev Dyn. 2005,233(1): 115-21.
[24] Celil A B, Hollinger J O, Campbell PG. Osx transcriptional regulation is mediated by additional pathways to BMP2/Smad signaling[J]. J Cell Biochem, 2005,95(3):518-28.
[25] Igarashi M, Kamiya N, Hasegawa M et al. Inductive effects of dexamethasone on the gene expression of Cbfal, Osterix and bone matrix proteins during differentiation of cultured primary rat osteoblasts[J]. J Mol Histol,2004,35 (1):3-10.
[26] Woodbury D, Schwarz E J, Prockop D J, et al. Adult rat and human bone marrow stromal cells differentiate into neurons[J]. Journal of Neuroscience Research,2000,61:364-370.
[27] Xudong LI,Quanjun Cui,Chinghai Kao, et al. Lavastatin inhibits adipogenic and stimulates osteogenic differentiation by suppressing PPAR_γ, and increasing Cbfa1/Runx2 expression in bone marrow mesenchymal cell cultures[J]. Bone, 2003,33:652-659.
[28] Miura M, Chen X D, Allen M R, et al. A crucial role of caspase-3 in osteogenic differentiation of bone marrow stromal stem cells[J]. J Clin Invest, 2004,114(12):1704-13.
[29] Bielby R C, Boccaccini A R, Polak JM,et al. In vitro differentiation and in vivo mineralization of osteogenic cells derived from human embryonic stem cells[J]. Tissue Eng, 2004, 10(9-10): 1518-25.[30] Reddi A H. Extracellular matrix and development[J].Extracellular Matrix Biochemistry ,1984,247-291.
[31] Reddi AH. Morphogenesis and tissue engineering of bone and cartilage: inductive signal,stem cell, and biomimetic biomaterials[J]. Tissue Engineering,2000,6(4):351-359.
[32] Ma S, Chen G, and Reddi A H. Collaboration between collagenous matrix and osteogenin is required for bone induction[J]. Ann.N.Y.Acad.Sci,1990,580:524-525.
[33] Ryan MP, William R, Huckle, et ah Effect of dexamethason withdrawal onosteoblastic differentiation of bone marrow stromal cells[J]. Journal of cellular biochemistry,2003,90:13-22.
[34] Otto F,Lubbert M,and Stock M. Upstream and downstream targets of Runx proteins[J]. Cell Biochem, 2003,89:9-18.
[35] Dong J, Uemura T, Kikuchi M, Tanaka J, et al. Long-term durability of porous hydroxyapatite with low-pressure system to support osteogenesis of mesenchymal stem cells[J]. Biomed Mater Eng, 2002;12(2):203-9.
[36] Nishimura H, Nishimura M, Oda R, Yamanaka K, et ah Lectins induce resistance to proteases and/or mechanical stimulus in all examined cells--including bone marrow mesenchymal stem cells--on various scaffolds[J]. Exp Cell Res, 2004 Apr 15;295(1):119-27.
[37] Laboureau J, Dubertret L, Lebreton D C, et al. ERK activation by mechanical strain is regulated by the small G proteins rac-1 and rhoA[J]. Exp.Derma, 2004.13:70-77.
[38] Ziros P G, Gil A P, Georgakopoulos T, et al. The bone-specific transcriptional regulator Cbfa1 is a target of mechanical signals in osteoblastic cells[J]. J Biol Chem, 2002,277(26):23934-41.
[39] Costessi A, Pines A, D'Andrea P, et al. Extracellular nucleotides activate Runx2 in the osteoblast-like HOBIT cell line: a possible molecular link between mechanical stress and osteoblasts' response[J]. Bone,2005 ,36(3):418-32.
[40] Altaian G H, Horan R L, Martin I, et al. Cell differentiation by mechanical stress. FASEB.J,2001,28:1096.
[41] Raab-Cullen D M, Thiede M A, Peterson D N, et al. Mechanical loading stimulates rapid changes in periosteal gen expression[J]. Calcif Tiss Int,1994,55:473-478.
[42] Inaoka T,Lean J M, Bessho T, et al. Sequential analysis of gene expression after an osteogenic stimulus:c-fos expression is induced in osteocytes[J]. Biochem Biophys Res Commun,1995,217:264-270.
[43] Wong M, Siegrist M, Goodwin K. Cyclic tensile strain and cyclic hydrostatic pressure differentially regulate expression of hypertrophic markers in primary chondrocytes[J]. Bone, 2003 ,33(4):685-93.[44] Wang M D. Manipulation of single molecules in biology[J]. Curr Opin Biotechnol,1999,10(1):81-6.
[45] Ahdjoudj S, Lasmoles F, Holy X, et al. Transforming growth factor beta2 inhibits adipocyte differentiation induced by skeletal unloading in rat bone marrow stroma[J]. J Bone Miner Res,2002, 17(4):668-77.
[46] Shigeo M T, Jiliang Li, Randall L, et al. Effects of broad frequency vibration on cultured osteoblasts[J].Journal of Biomechanics,2003,36:73-80.
[47] Karmo T, Takahashi T, Ariyoshi W, et al. Tensile mechanical strain up-regulates Runx2 and osteogenic factor expression in human periosteal cells: implications for distraction osteogenesis[J]. J Oral Maxillofac Surg. 2005,63(4):499-504.
[48] Ignatius A, Blessing H, Liedert A, et al. Effects of mechanical strain on human osteoblastic precursor cells in type I collagen matrices[J]. Orthopade. 2004,33(12): 1386-93.
[49] Koike M, Shimokawa H, Kanno Z et al. Effects of mechanical strain on proliferation and differentiation of bone marrow stromal cell line ST2[J]. J Bone Miner Metab, 2005;23(3):219-25.
[50] 翟中和，王喜忠，丁明孝，主编．细胞生物学[M]．北京：高等教育出版社，2000，319-343．
[51] Wang N, Ingber D E. Control of cytoskeletal mechanics by extracellular matrix, cell shape, and mechanical tension[J]. Biophysical Journal, 1994,66:2181-2189.
[52] Jennifer S P, Julia S F, Catherine Cheng et al. Differential effects of equiaxial and uniaxial strain on mesenchymal stem cells[J]. Biotechnology and Bioengineering, 2004,88 (3):360-368.
[53] Wang N,Vujak-Novakovik G.Mechanotransduction across the cell surface and through the cytoskeleton[J]. Science, 1993, 260:1124-17.
[54] Meazzini M C,Toma C D,Schaffer I L,et al.Ostoeblast cytoskeletal modulation in response to mechanical strain in vitro[J]. J Orthop Res, 1998, 16(2)170-80.
[55] 杨美祥，林珠，丁寅等．不同应力对人牙周膜成纤维细胞细胞骨架影响的体外研究[J]．中国临床康复，20013，7(23)：3162-63．
[56] Banneyx G, Baugh L, Vogel V. Fibronectin extension and unfolding with in cell matrix fibrils controlled by cytoskeletal tension[J]. Proc Natl Acad Sci USA, 2002, 99(8):5139-43.
[57] Takemasa T, Sugimoto K,Yamashita K. Amplitude-dependent stress fiber reorientation in early response to cyclic strain[J]. Experimental Cell Research, 1997.77:91-99.
[58] 霍霞，吕建勋，杨仁东等．早期人羊膜共聚焦激光扫描光学切片及F-肌动蛋白的研究[J]．解剖学报，2002，33(6)：283-87．
[59] Dartsch P C, Betz E. Response of cultured endothelial cells to mechanical stimulation[J]. Basic Research in Cardiology, 1989, 84:268-281.[60] James H C, Pascal G C, Jeremiah Wille, et al. Specificity of endothelial cell reorientation in response to cyclic mechanical stretching[J].Journal of Biomechanics,2001,34:1563-1572.
[61] 刘景生 主编．细胞信息与调控{M}．北京医科大学、中国协和医科大学联合出版社，北京．187-200
[62] Duncan R L, Hatter L V, Levin D W, et al. Regulation of strech-activated cation channel activity via the cytoskeleton and similar to hormonal regulation. Mol Biol Cell, 1992,3:38a.
[63] Cheng H,Lederer W J,Cannel M B. Calcium sparks:elementary events underlying excitation-contraction coupling in heart muscle[J].science,1993,262:740-744.
[64] Isaeva E V, Shkryl VM,Shirokova N. Mitochondrial redox state and Ca~(2+) sparks in permeabilized mammalian, skeletal muscle[J].J Physiol,2005,565:855-872.
[65] Kamishima T, Quayle J M. Ca2+-induced Ca~(2+) release in cardiac and smoothmucclecells.BiochemicalSocietyTransactions,2003,31:943-946
[66] Guharay F, Sashs F. Stretch-activated single ion channel current in tissue cultured embryonic chick skeletal muscle[J]. 1984,352:685-701.
[67] Petkov G V, Nelson M T. Differential regulation of Ca2+-activated K+ channels by beta-adrenoceptors in guinea pig urinary bladder smooth muscle[J].Am J Physiol Cell Physiol,2005,288(6):C1255-63.
[68] Walker L M,Publicover S J,Preston M R, et al. Calcium-channel activation and matrix protein upregulation in bone cells in response to mechanical strain[J].Joumal of Cellular Biochemistry,2000,79:648-661.
[69] Xia S L, Ferrier J. Calcium signal induced by mechanical perturbation of osteoclasts[J].J Cell Physiol, 1995,163:493-501.
[70] Zaman G;Suswillo RFL,Cheng M Z, et al. Early responses to dynamic strain changes and prostaglandins in bone-derived cells in culture[J]. J Bone Miner Res ,1997,12:132-136.
[71] Li X, Zima A V, Sheikh F, et al. Endothelin-1-induced arrhythrnogenic Ca~(2+) signaling is abolished in atrial myocytes of inositol-1,4,5- trisphosphateTP3)-receptor type 2-deficient mice[J].Circ Res, 2005, 96(12): 1:274-81.
[72] Chandler W.K, Hollingworth S and S.M. Baylor. Simulation of Calcium Sparks in Cut Skeletal Muscle Fibers of the Frog[J]. Journal of General Physiology, 2003,121 (4):311-324.
[73] 张钦，程和平．心肌细胞钙火花[J]．中国药理学通报．2005，21(1)：23-26．
[74] Zhou J, Brum G, Gonzalez A, et al. Ca2+ sparks and embers of mammalian muscle. Properties of the sources[J] .J Gen Physiol, 2003,122(1):95-114
[75] Inone M,and Bridge J H. Ca2+ sparks in rabbit ventricular myocytes evoked by action potentials: involvement of clusters of L-type Ca2+ channels[J]. Circ Res,2003,92(5):532-8.[76] Jessop H L,and Rawlinson S C, Pitsillides AA et al. Mechanical strain and fluid movement both activate extracellular regulated kinase(ERK) in osteoblast like cells but via different signaling pathways. Bone, 2002,31:186-194.
[77] Long.P, Liu.F, Piesco.N P, et al. 2002.Signaling by mechanical strain involves transcriptional regulation of proinflammatory genes in human periodontal ligament cellsin vitro. Bone,30:547-552.
[78] Laboureau J,Dubertret L, Lebreton D C, et al. ERK activation by mechanical strain is regulated by the small G proteins rac-1 and rhoA. Exp.Derma, 2004,13:70-77.
[79] Kanno T, Takahashi T, Ariyoshi W, et al. Tensile mechanical strain up-regulates Runx2 and osteogenic factor expression in human periosteal cells: implications for distraction osteogenesis[J]. J Oral Maxillofac Surg, 2005 ,63(4):499-504.
[80] Liang F, and Gardner D G Mechanical strain activates BNP gene transcription through a p38/NF-kappa-B-dependent mechanism[J]J.Clin. Invest, 1999.104,1603-1612.
[81] Carey M B,and Matsumoto S GCalcium transient activity in culured murine neural crest cells is regulated at IP3 receptor[J] .Brain Research,2000,862:201-210.
[82] Carpio L,Gladu J,Goltzman D et al. Induction of osteoblast differentiation indexes by PTHrP in MG-63 cells involves multiple signaling pathways[J]. Am J Physiol Endocrinol Metab, 2001,281:E489-99.
[83] Jessop H L, Rawlinson S C, Pitsillides AA,et al. Mechanical strain and fluid movement both activate extracellular regulated kinase(ERK) in osteoblast like cells but via different signaling pathways[J]. Bone, 2002,31:186-194.
[84] Berridge MJ. The biology and medicin of calcium signaling. Mol Cellular Endocri,1994,98:119-1