人细胞外基质磷酸化糖蛋白的重组表达及在牙本质形成中的作用和机理研究
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摘要
MEPE,又名成骨细胞/骨细胞因子45(Osteoblast/Osteocyte Factor 45;OF45)或是Osteoregulin。2000年,Rowe在筛选肿瘤性低血磷性骨软化症(oncogenic hypophosphatemic osteomalacia,OHO)患者肿瘤组织cDNA文库中表达上调的基因时,发现了MEPE。RT-PCR检测发现,正常组织中,MEPE在骨髓和大脑中表达,肺、肾和胎盘中的表达水平很低。同年,Petersen等在大鼠骨髓源性的成骨细胞中克隆到MEPE基因,Northern blot和免疫组织化学研究显示:MEPE仅在骨组织中表达,特别是骨小梁和皮质骨中的骨细胞呈高表达。2003年,Gowen等克隆了小鼠MEPE基因,发现其上游存在三个蛋氨酸密码子,可以选择性转录起始,最大可编码441个氨基酸残基的蛋白质。Northern和Western blot显示MEPE在骨组织和成牙本质细胞中表达。目前在口腔医学领域MEPE的研究还是处于初期,对于MEPE的功能我们还知之甚少。前期研究提示MEPE可能参与牙本质形成。本课题通过MEPE基因克隆、原核以及真核蛋白表达等方法,在mRNA和蛋白质的水平上,从MEPE的表达、在牙齿细胞中功能以及在羟基磷灰石晶体形成和生长中作用等几个方面,全面深入地研究MEPE参与牙本质基质形成和矿化的过程、作用和机理等。这将阐明MEPE在牙本质牙髓复合体形成和损伤修复中的机制,丰富牙髓生物学和牙齿发育生物学,并为临床上生活牙髓的保存治疗和组织工程化牙本质修复牙体组织缺损提供理论基础
本研究分为以下5个部分内容:
一、MEPE基因克隆和序列分析
利用PCR技术,设计一对引物从人脑cDNA文库中扩增出MEPE编码区基因片段(约1.5kb),将扩增的基因片段插入pGEM-T Easy克隆载体,限制性酶切进行鉴定并进行核苷酸序列测定。结果表明:克隆到人MEPE蛋白编码区基因片段,序列与GenBank中收录的人MEPE cDNA序列一致。
二、MEPE基因在原核和真核细胞中的表达
将MEPE编码区全长片段亚克隆至表达载体pGEX-4T-1中,大肠杆菌中诱导表达,纯化后获得人MEPE蛋白。以重组、纯化的MEPE为抗原,混入完全/不完全弗氏佐剂,免疫新西兰大白兔,制备MEPE多克隆抗体。
分别将MEPE基因亚克隆入非融合和融合真核表达载体pcDNA3.1和pEGFP-C3中,脂质体介导将载体转染哺乳动物细胞COS-7,通过免疫组织化学、Western Blot等方法检测MEPE蛋白在哺乳动物细胞中的表达。发现表达的绿色荧光蛋白融合蛋白所发出的荧光位于细胞浆中,胞核中无荧光,与免疫组化结果一致,说明MEPE位于细胞浆中。Western Blot检测显示在COS-7细胞中表达的MEPE的分子量为61kDa,与理论值相符。
三、MEPE蛋白的组织表达特性研究
用制备的抗人MEPE蛋白抗体对狗牙胚组织切片进行免疫组化染色,发现成牙本质细胞染色阳性,前期牙本质染色阳性,牙髓细胞染色阴性。
四、MEPE蛋白对牙髓细胞和牙周膜细胞的生物学作用
采用四唑盐比色法观察了MEPE蛋白对人牙周膜细胞、牙髓细胞增殖的影响,结果发现MEPE蛋白对牙周膜细胞增殖无促进作用、对牙髓细胞的增殖有促进作用。酶动力学方法观察了MEPE蛋白对人牙周膜细胞、牙髓细胞ALP活性的影响。研究结果表明,MEPE蛋白可提高牙髓细胞、牙周膜细胞的ALP活性。Von kossa染色结果显示MEPE蛋白可促进牙髓细胞的体外矿化。
五、采用稳态琼脂糖凝胶的HA生长系统,研究MEPE蛋白质在体外矿化中的作用
本实验首次将不同浓度的MEPE蛋白质加入HA晶体生长系统的平衡透析池,测定凝胶中磷酸盐和钙离子浓度、以及HA晶体的大小和完整性,证明MEPE蛋白质在体外可以促进HA晶体形成、生长。
Recently, a new bone matrix protein cDNA has been cloned from human, rat, and mouse by independent groups . The human clone, termed MEPE(matrix extracellular phosphoglycoprotein), was isolated from a human oncogenic hypophosphatemic osteomalacia tumor(OHO) cDNA library with polyclonal antibodies that neutralized OHO tumor secreted phosphate uptake-inhibiting factor . MEPE , BSP (Bone sialoprotein), OPN (osteopontin, but sometimes known as SPP1 and Eta-1), DMP I(dentin matrix protein I), DSPP(dentin sialophosphoprotein) are the products of five genes clustered along human chromosome 4q21 between EST markers D4S2785(WI-6336) and D4S2844. The majority of each protein is encoded by the last one or two exons and contains the integrin-binding RGD tripeptide. Another point of similarity of all these genes is that all introns always interrupt between codons (type 0), thus leaving open the possibility of splicing together any two exons without causing frame shifts. We have named this family of proteins the SIBLING family for Small Integrin-Binding LIgand, N-linked Glycoprotein based not on current theories of their functions (which are poorly understood) but based on the simple biochemical and genetic features shared by all members. The human MEPE has 1989 bp cDNA clone and encodes a predicted 525-amino-acid protein rich in Asp, Ser, and Glu residues (26%) containing a 17-amino-acid signal peptide. MEPE contains two N-glycosylation motifs (NNSTandNNSR),a glycosaminoglycan attachment site (SGDG), an RGD cell attachment motif, several predicted phosphorylation motifs,and N-myristoylation sites.
In dental tissue, MEPE is expressed in odontoblasts during odontogenesis, Liu has found that Dentonin(a 23-amino-acid peptide derived from MEPE) can promote DPSC proliferation, taking a potential role in pulp repair. That study suggestted that Dentonin affects primarily the innitial cascade of events leading to pulp healing.
In this study, we have cloned MEPE cDNA by RT-PCR, expressed MEPE in E.coli and COS-7 cells, purified the recombinant protein, prepared anti-MEPE antibodies. Then we proceeded western blot and immunohistochemistry. Finally, a Steady-State Agarose Gels HA Growth System will be used to study the effects of MEPE to HA.
The matrix extracellular phosphoglycoprotein (MEPE) gene is highly expressed in tumors that cause onco-genic hypophosphatemic osteomalacia (OHO). MEPE is also known as one of the bone-tooth matrix proteins and is associated with bone and teeth mineralization. We developed a rabbit polyclonal antibody directed against recombinant human MEPE after cloning its cDNA from the cDNA library of a human brain cDNA library the predicted secreted MEPE protein has a calculated molecular mass of 56 kDa. Like human MEPE, Fourteen of the serine residues in Mepe are potential phosphorylation sites for protein kinase C, casein kinase II, and cAMP-dependent protein kinase. As reported for human MEPE, a specific feature of the MEPE protein is the occurrence in the C-terminus of a serine-rich sequence, DDSSESSDSGSSSES (residues 417–430), that displays homology to repeat motifs found in dentin phosphoprotein, DPP/DSPP (SDSSDSSDSSSSSDSS), DMP1 (SSRRRDDSSESSDSGSSSESDG), osteopontin (DDSHQSDESHHSDESD) and the parent protein, dentin sialophosphoprotein,.. Using this anti-body, we analyzed the distribution of MEPE in dog dental germ tissure by immunohistochemistry. In this specimens, MEPE was predominantly expressed by odontoblasts cell and predentin not by dental pulp cells. Furthermore we use a steady-state agarose gel system, MEPE were incorporated at 5-25μg/ml into steady-state agarose gels and incubated for 5 days, MEPE caused significantly higher levels of calcium phosphate accumulation than control gels didthe results reported here suggest that MEPE can induce HA and we propose that this protein has a potential effect on dental rehabilitation. .
本研究分为以下5个部分内容:
一、MEPE基因克隆和序列分析
利用PCR技术,设计一对引物从人脑cDNA文库中扩增出MEPE编码区基因片段(约1.5kb),将扩增的基因片段插入pGEM-T Easy克隆载体,限制性酶切进行鉴定并进行核苷酸序列测定。结果表明:克隆到人MEPE蛋白编码区基因片段,序列与GenBank中收录的人MEPE cDNA序列一致。
二、MEPE基因在原核和真核细胞中的表达
将MEPE编码区全长片段亚克隆至表达载体pGEX-4T-1中,大肠杆菌中诱导表达,纯化后获得人MEPE蛋白。以重组、纯化的MEPE为抗原,混入完全/不完全弗氏佐剂,免疫新西兰大白兔,制备MEPE多克隆抗体。
分别将MEPE基因亚克隆入非融合和融合真核表达载体pcDNA3.1和pEGFP-C3中,脂质体介导将载体转染哺乳动物细胞COS-7,通过免疫组织化学、Western Blot等方法检测MEPE蛋白在哺乳动物细胞中的表达。发现表达的绿色荧光蛋白融合蛋白所发出的荧光位于细胞浆中,胞核中无荧光,与免疫组化结果一致,说明MEPE位于细胞浆中。Western Blot检测显示在COS-7细胞中表达的MEPE的分子量为61kDa,与理论值相符。
三、MEPE蛋白的组织表达特性研究
用制备的抗人MEPE蛋白抗体对狗牙胚组织切片进行免疫组化染色,发现成牙本质细胞染色阳性,前期牙本质染色阳性,牙髓细胞染色阴性。
四、MEPE蛋白对牙髓细胞和牙周膜细胞的生物学作用
采用四唑盐比色法观察了MEPE蛋白对人牙周膜细胞、牙髓细胞增殖的影响,结果发现MEPE蛋白对牙周膜细胞增殖无促进作用、对牙髓细胞的增殖有促进作用。酶动力学方法观察了MEPE蛋白对人牙周膜细胞、牙髓细胞ALP活性的影响。研究结果表明,MEPE蛋白可提高牙髓细胞、牙周膜细胞的ALP活性。Von kossa染色结果显示MEPE蛋白可促进牙髓细胞的体外矿化。
五、采用稳态琼脂糖凝胶的HA生长系统,研究MEPE蛋白质在体外矿化中的作用
本实验首次将不同浓度的MEPE蛋白质加入HA晶体生长系统的平衡透析池,测定凝胶中磷酸盐和钙离子浓度、以及HA晶体的大小和完整性,证明MEPE蛋白质在体外可以促进HA晶体形成、生长。
Recently, a new bone matrix protein cDNA has been cloned from human, rat, and mouse by independent groups . The human clone, termed MEPE(matrix extracellular phosphoglycoprotein), was isolated from a human oncogenic hypophosphatemic osteomalacia tumor(OHO) cDNA library with polyclonal antibodies that neutralized OHO tumor secreted phosphate uptake-inhibiting factor . MEPE , BSP (Bone sialoprotein), OPN (osteopontin, but sometimes known as SPP1 and Eta-1), DMP I(dentin matrix protein I), DSPP(dentin sialophosphoprotein) are the products of five genes clustered along human chromosome 4q21 between EST markers D4S2785(WI-6336) and D4S2844. The majority of each protein is encoded by the last one or two exons and contains the integrin-binding RGD tripeptide. Another point of similarity of all these genes is that all introns always interrupt between codons (type 0), thus leaving open the possibility of splicing together any two exons without causing frame shifts. We have named this family of proteins the SIBLING family for Small Integrin-Binding LIgand, N-linked Glycoprotein based not on current theories of their functions (which are poorly understood) but based on the simple biochemical and genetic features shared by all members. The human MEPE has 1989 bp cDNA clone and encodes a predicted 525-amino-acid protein rich in Asp, Ser, and Glu residues (26%) containing a 17-amino-acid signal peptide. MEPE contains two N-glycosylation motifs (NNSTandNNSR),a glycosaminoglycan attachment site (SGDG), an RGD cell attachment motif, several predicted phosphorylation motifs,and N-myristoylation sites.
In dental tissue, MEPE is expressed in odontoblasts during odontogenesis, Liu has found that Dentonin(a 23-amino-acid peptide derived from MEPE) can promote DPSC proliferation, taking a potential role in pulp repair. That study suggestted that Dentonin affects primarily the innitial cascade of events leading to pulp healing.
In this study, we have cloned MEPE cDNA by RT-PCR, expressed MEPE in E.coli and COS-7 cells, purified the recombinant protein, prepared anti-MEPE antibodies. Then we proceeded western blot and immunohistochemistry. Finally, a Steady-State Agarose Gels HA Growth System will be used to study the effects of MEPE to HA.
The matrix extracellular phosphoglycoprotein (MEPE) gene is highly expressed in tumors that cause onco-genic hypophosphatemic osteomalacia (OHO). MEPE is also known as one of the bone-tooth matrix proteins and is associated with bone and teeth mineralization. We developed a rabbit polyclonal antibody directed against recombinant human MEPE after cloning its cDNA from the cDNA library of a human brain cDNA library the predicted secreted MEPE protein has a calculated molecular mass of 56 kDa. Like human MEPE, Fourteen of the serine residues in Mepe are potential phosphorylation sites for protein kinase C, casein kinase II, and cAMP-dependent protein kinase. As reported for human MEPE, a specific feature of the MEPE protein is the occurrence in the C-terminus of a serine-rich sequence, DDSSESSDSGSSSES (residues 417–430), that displays homology to repeat motifs found in dentin phosphoprotein, DPP/DSPP (SDSSDSSDSSSSSDSS), DMP1 (SSRRRDDSSESSDSGSSSESDG), osteopontin (DDSHQSDESHHSDESD) and the parent protein, dentin sialophosphoprotein,.. Using this anti-body, we analyzed the distribution of MEPE in dog dental germ tissure by immunohistochemistry. In this specimens, MEPE was predominantly expressed by odontoblasts cell and predentin not by dental pulp cells. Furthermore we use a steady-state agarose gel system, MEPE were incorporated at 5-25μg/ml into steady-state agarose gels and incubated for 5 days, MEPE caused significantly higher levels of calcium phosphate accumulation than control gels didthe results reported here suggest that MEPE can induce HA and we propose that this protein has a potential effect on dental rehabilitation. .
引文
1 Butler WT, Ritchie H. The nature and functional significance of dentin extracellular matrix proteins. Int J Dev Biol.1995;39(1):169
2 Xiao S, Yu C, Chou X, Yuan W, Wang Y, Bu L, Fu G, Qian M, Yang J, Shi Y, Hu L, Han B, Wang Z, Huang W, Liu J, Chen Z, Zhao G, Kong X.. Dentinogenesis imperfecta 1 with or without progressive hearing loss is associated with distinct mutations in DSPP. Nat Genet,2001;27(2):201
3 于世风.口腔组织病理学.第 4 版.北京:人民卫生出版社,2000.84-85.
4 Mitsiadis TA, Roméas A, Lendahl U, Sharpe PT, Farges JC. Notch2 protein distribution in human teeth under normal and pathological conditions. Exp Cell Res. 2003;282(2):101-9.
5 金岩.口腔颌面组织胚胎学.陕西:陕西科学技术出版社,2002.110-131.
6 Ruch JV. Odontoblast commitment and differentiation. Biochem Cell Biol. 1998; 76(6): 923-38.
7 Mjar IA, Sveen OB, Heyeraas KJ. Pulp-dentin biology in restorative dentistry. Part 2: initial reactions to preparation of teeth for restorative procedures. Quintessence Int. 2001; 32(7):537-51.
8 Ruch JV, Lesot H, Bègue-Kirn C. Odontoblast differentiation. Int J Dev Biol. 1995; 39(1): 51-68.
9 邓蔓菁,金岩,史俊南.诱导成牙本质样细胞在体内外的矿化研究.牙体牙髓牙周病学杂志,2003,13:423-425.
10 Unda FJ, Martín A, Hernandez C, Pérez-Nanclares G, Hilario E, Aréchaga J. FGFs-1 and -2, and TGF beta 1 as inductive signals modulating in vitro odontoblast differentiation. Adv Dent Res. 2001; 15:34-7.
11 于世风.口腔组织病理学.第 4 版.北京:人民卫生出版社,2000.84-85.
12 Arana-Chavez VE, Katchburian E. Freeze-fracture studies of the distal plasma membrane of rat odontoblasts during their differentiation and polarisation. Eur J Oral Sci. 1998; 106(Suppl1): 132-6.
13 Sasagawa I. Mineralization patterns in elasmobranch fish. Microsc Res Tech. 2002; 59(5): 396-407.
14 Linde A. Dentin mineralization and the role of odontoblasts in calcium transport. Connect Tissue Res. 1995; 33(1-3): 163-70.
15 Butler WT. Dentin matrix proteins. Eur J Oral Sci. 1998; 106 (Suppl1): 204-10.
16 Boskey A, Spevak L, Tan M, Doty SB, Butler WT. Dentin sialoprotein (DSP) has limited effects on in vitro apatite formation and growth. Calcif Tissue Int. 2000; 67(6): 472-8.
17 Narayanan K, Srinivas R, Ramachandran A, Hao J, Quinn B, George A. Differentiation of embryonic mesenchymal cells to odontoblast-like cells by overexpression of dentin matrix protein 1. Proc Natl Acad Sci U S A. 2001; 98(8): 4516-21.
18 Thyagarajan T, Sreenath T, Cho A, Wright JT, Kulkarni AB. Reduced expression of dentin sialophosphoprotein is associated with dysplastic dentin in mice overexpressing transforming growth factor-beta 1 in teeth. J Biol Chem. 2001; 276(14):11016-20.
19 Goldberg M, Smith AJ. Cells and extracellular matrices of dentin and pulp: a biological basis for repair and tissue engineering. Crit Rev Oral Biol Med. 2004; 15(1): 13-27.
20 Chai Y, Slavkin HC. Prospects for tooth regeneration in the 21st century: a perspective. Microsc Res Tech. 2003; 60(5):469-79.
21 Ayanoglou CM, Godeau G, Lesty C, Septier D, Goldberg M. CyclosporinA-induced alterations of dentinogenesis in rat molars. J Oral Pathol Med. 1997; 26(3): 129-34.
22 Pekkala E, V?likangas L, Puukka M, Tj?derhane L, Larmas M. The effect of a high-sucrose diet on dentin formation and dental caries in hyperinsulinemic rats. J Dent Res. 2002; 81(8): 536-40.
23 Leonora J, Tj?derhane L, Tieche JM. Parotid gland function and dentin apposition in rat molars. J Dent Res. 2002; 81(4): 259-64.
24 Rowe PS, de Zoysa PA, Dong R, Wang HR, White KE, Econs MJ, Oudet CL. MEPE, a new gene expressed in bone marrow and tumors causing osteomalacia. Genomics.2000;67(1):54
25 Petersen DN, Tkalcevic GT, Mansolf AL, Rivera-Gonzalez R, Brown TA. Identification of osteoblast/osteocyte factor 45 (OF45), a bone-specific cDNA encoding an RGD-containing protein that is highly expressed in osteoblasts and osteocytes. J Biol Chem. 2000;275(46):36172
26 Argiro L, Desbarats M, Glorieux FH, Ecarot B. Mepe, the gene encoding a tumor-secreted protein in oncogenic hypophosphatemic osteomalacia, is expressed in bone. Genomics. 2001;74(3):342.
27 Bresler D, Bruder J, Mohnike K, Fraser WD, Rowe PS. Serum MEPE- ASARM-peptides are elevated in X-linked rickets (HYP): implications for phosphaturia and rickets. J Endocrinol. 2004;183(3):R1
28 Rowe PS, Garrett IR, Schwarz PM, Carnes DL, Lafer EM, Mundy GR, Gutierrez GE. Surface plasmon resonance (SPR) confirms that MEPE binds to PHEX via the MEPE-ASARM motif: a model for impaired mineralization in X-linked rickets (HYP). Bone. 2005; 36(1):33-46.
29 Hayashibara T, Hiraga T, Yi B, Nomizu M, Kumagai Y, Nishimura R, Yoneda T. A synthetic peptide fragment of human MEPE stimulates new boneformation in vitro and in vivo. J Bone Miner Res. 2004; 19(3):455-62.
30 Liu H, Li W, Gao C, Kumagai Y, Blacher RW, DenBesten PK. Dentonin, a fragment of MEPE, enhanced dental pulp stem cell proliferation. J Dent Res. 2004;83(6):496
31 Gowen LC, Petersen DN, Mansolf AL, Qi H, Stock JL, Tkalcevic GT, Simmons HA, Crawford DT, Chidsey-Frink KL, Ke HZ, McNeish JD, Brown TA. Targeted disruption of the osteoblast/osteocyte factor 45 gene (OF45) results in increased bone formation and bone mass. J Biol Chem. 2003;278(3):1998-2007
32 Liu S, Brown TA, Zhou J, Xiao ZS, Awad H, Guilak F, Quarles LD. Role of matrix extracellular phosphoglycoprotein in the pathogenesis of X-linked hypophosphatemia. J Am Soc Nephrol. 2005;16(6):1645-53.
33 Qin C, Baba O, Butler WT. Post-translational modifications of sibling proteins and their roles in osteogenesis and dentinogenesis. Crit Rev Oral Biol Med. 2004;15(3):126
34 Fisher LW, Fedarko NS. Six genes expressed in bones and teeth encode the current members of the SIBLING family of proteins. Connect Tissue Res. 2003; 44 (Suppl 1):33-40.
35 White KE, Lorenz B, Evans WE, Meitinger T, Strom TM, Econs MJ. Molecular cloning of a novel human UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferase, GalNAc-T8, and analysis as a candidate autosomal dominant hypophosphatemic rickets (ADHR) gene. Gene. 2000; 246(1-2): 347-56.
36 Quarles LD, Drezner MK. Pathophysiology of X-linked hypophosphatemia, tumor-induced osteomalacia, and autosomal dominant hypophosphatemia: a perPHEXing problem. J Clin Endocrinol Metab. 2005; 86(2): 494-6.37 Shimada T, Mizutani S, Muto T, Yoneya T, Hino R, Takeda S, Takeuchi Y, Fujita T, Fukumoto S, Yamashita T. Cloning and characterization of FGF23 as a causative factor of tumor-induced osteomalacia. Proc Natl Acad Sci U S A. 2001; 98(11): 6500-5.
38 Murer H, Forster I, Hernando N, Lambert G, Traebert M, Biber J. Posttranscriptional regulation of the proximal tubule NaPi-II transporter in response to PTH and dietary P(i). Am J Physiol. 1999; 277(5 Pt 2): F676-84.
39 Bacic D, Wagner CA, Hernando N, Kaissling B, Biber J, Murer H. Novel aspects in regulated expression of the renal type IIa Na/Pi-cotransporter. Kidney Int Suppl. 2007; (91):S5-S12.
40 Siggelkow H, Schmidt E, Hennies B, Hüfner M. Evidence of downregulation of matrix extracellular phosphoglycoprotein during terminal differentiation in human osteoblasts. Bone. 2004; 35(2):570-6.
41 Liu H, Li W, Shi S, Habelitz S, Gao C, Denbesten P. MEPE is downregulated as dental pulp stem cells differentiate. Arch Oral Biol. 2005; 50(11):923-8.
42 Igarashi M, Kamiya N, Ito K, Takagi M. In situ localization and in vitro expression of osteoblast/osteocyte factor 45 mRNA during bone cell differentiation. Histochem J. 2002; 34(5): 255-63.
43 Nampei A, Hashimoto J, Hayashida K, Tsuboi H, Shi K, Tsuji I, Miyashita H, Yamada T, Matsukawa N, Matsumoto M, Morimoto S, Ogihara T, Ochi T, Yoshikawa H. Matrix extracellular phosphoglycoprotein (MEPE) is highly expressed in osteocytes in human bone. J Bone Miner Metab. 2004; 22(3):176-84.
44 Guo R, Rowe PS, Liu S, Simpson LG, Xiao ZS, Quarles LD. Inhibition of MEPE cleavage by Phex. Biochem Biophys Res Commun. 2002; 297(1):38-45.
45 Liu S, Guo R, Simpson LG, Xiao ZS, Burnham CE, Quarles LD. Regulation of fibroblastic growth factor 23 expression but not degradation by PHEX. J Biol Chem. 2006; 278(39): 37419-26.
46 Quarles LD. Evidence for a bone-kidney axis regulating phosphate homeostasis. J Clin Invest. 2003; 112(5): 642-6.
47 Lu C, Huang S, Miclau T, Helms JA, Colnot C. Mepe is expressed during skeletal development and regeneration. Histochem Cell Biol. 2004; 121(6): 493-9.
48 Jain A, Fedarko NS, Collins MT, Gelman R, Ankrom MA, Tayback M, Fisher LW. Serum levels of matrix extracellular phosphoglycoprotein (MEPE) in normal humans correlate with serum phosphorus, parathyroid hormone and bone mineral density. J Clin Endocrinol Metab. 2004; 89(8): 4158-61.
49 Nagel DE, Khosla S, Sanyal A, Rosen DM, Kumagai Y, Riggs BL. A fragment of the hypophosphatemic factor, MEPE, requires inducible cyclooxygenase-2 to exert potent anabolic effects on normal human marrow osteoblast precursors. J Cell Biochem. 2004; 93(6): 1107-14.
50 胡宝成,薛恒钢,刘爽. 一种新的抗辐射性相关蛋白 AA12 和 CHK1 相互作用的研究.军事医学科学院院刊,2003; 27(5): 345-348.
51 Smits VA, Medema RH. Checking out the G(2)/M transition. Biochim Biophys Acta. 2001; 1519(1-2): 1-12.
52 Khanna KK. Cancer risk and the ATM gene: a continuing debate. J Natl Cancer Inst. 2005; 92(10):795-802.
53 Zoe A, Stewart L, Jennifer A. Cell cycle checkpoints as therapeu tic targets. J Mammary Gland Biol Neoplasia,1999; 4(4): 389-400.
54 Martinez J, Georgoff I, Martinez J, Levine AJ. Cellular localization and cell cycle regulation by a temperature-sensitive p53 protein. Genes Dev. 1991; 5(2):151-9.
55 Hu B, Zhou XY, Wang X, Zeng ZC, Iliakis G, Wang Y. The radioresistance to killing of A1-5 cells derives from activation of the Chk1 pathway. J Biol Chem. 2001; 276(21): 17693-8.
56 胡宝成, 王翔, Iliakis G. 转化的大鼠胚胎成纤维细胞系差异表达基因的筛选研究. 生物技术通讯. 2002;13(2): 118-125.
57 Holm IA, Nelson AE, Robinson BG, Mason RS, Marsh DJ, Cowell CT, Carpenter TO. Mutational analysis and genotype-phenotype correlation of the PHEX gene in X-linked hypophosphatemic rickets. J Clin Endocrinol Metab. 2001; 86(8): 3889-99.
58 Sabbagh Y, Boileau G, DesGroseillers L, Tenenhouse HS. Disease-causing missense mutations in the PHEX gene interfere with membrane targeting of the recombinant protein. Hum Mol Genet. 2005; 10(15): 1539-46.
59 MacDougall M, Simmons D, Gu TT, Dong J. MEPE/OF45, a new dentin/bone matrix protein and candidate gene for dentin diseases mapping to chromosome 4q21. Connect Tissue Res. 2002; 43(2-3): 320-30.
60 Beattie ML, Kim JW, Gong SG, Murdoch-Kinch CA, Simmer JP, Hu JC. Phenotypic variation in dentinogenesis imperfecta/dentin dysplasia linked to 4q21. J Dent Res. 2006; 85(4): 329-33.
61 金冬雁,黎孟枫,译.分子克隆实验指南.第二版.北京:科学出版社.1996
62 颜子颖,王海林,译.精编分子生物学实验指南.北京:科学出版社.1998
63 司徒镇强,吴军正.细胞培养.西安:世界图书出版社西安公司.1996;186
64 Quarles LD, Yohay DA, Lever LW, et al. Distinct proliferative and differentiated stages of murine MC3T3-E1 cells in culture: an in vitro model of osteoblast development. J Bone Miner Res. 1992; 7(6): 683-87
65 Pavlin D, Dove SB, Zadro R, et al. Mechanical loading stimulatesdifferentiation of periodontal osteoblasts in a mouse osteoinduction model: effect on type I collagen and alkaline phosphatase genes. Calcif Tissue Int. 2000; 67(2):163-72.
66 吴奇光.口腔组织病理学.北京:人民卫生出版社.1993;70
67 Robey PG. Bone matrix proteoglycans and glycoprotein. Principles of Bone Biology. Academic Press, San Diego,Ca,USA, 1996;155
68 Horiuch K, Amizuka N, Takeshita S. Identification and Characterization of a noval protein, Periostin, with restricted expression to periosteum and periodontal ligament and increased expression by transforming growth factor β. J Bone Miner Res. 1999;14:1239
69 Hunter GK, Goldberg HA. Nucleation of hydroxyapatite by bone sialoprotein. Proc Natl Acad Sci U S A,1993;90:8562
70 颜子颖,王海林,译.精编分子生物学实验指南.北京:科学出版社.1998
71 Kaufman RJ. Overview of Vectors used for expression in mammalian cells. Methods enzymol. 1990; 185: 487-527
72 Sampath TK, Maliakal JC, Hauschka PV et al. Recombinant human osteogenic protein-1 (hOP-1) induces new bone formation in vivo with a specific activity comparable with natural bovine osteogenic protein and stimulates osteoblast proliferation and differentiation in vitro. J Biol Chem 1992 ;267(28):20352-62
2 Xiao S, Yu C, Chou X, Yuan W, Wang Y, Bu L, Fu G, Qian M, Yang J, Shi Y, Hu L, Han B, Wang Z, Huang W, Liu J, Chen Z, Zhao G, Kong X.. Dentinogenesis imperfecta 1 with or without progressive hearing loss is associated with distinct mutations in DSPP. Nat Genet,2001;27(2):201
3 于世风.口腔组织病理学.第 4 版.北京:人民卫生出版社,2000.84-85.
4 Mitsiadis TA, Roméas A, Lendahl U, Sharpe PT, Farges JC. Notch2 protein distribution in human teeth under normal and pathological conditions. Exp Cell Res. 2003;282(2):101-9.
5 金岩.口腔颌面组织胚胎学.陕西:陕西科学技术出版社,2002.110-131.
6 Ruch JV. Odontoblast commitment and differentiation. Biochem Cell Biol. 1998; 76(6): 923-38.
7 Mjar IA, Sveen OB, Heyeraas KJ. Pulp-dentin biology in restorative dentistry. Part 2: initial reactions to preparation of teeth for restorative procedures. Quintessence Int. 2001; 32(7):537-51.
8 Ruch JV, Lesot H, Bègue-Kirn C. Odontoblast differentiation. Int J Dev Biol. 1995; 39(1): 51-68.
9 邓蔓菁,金岩,史俊南.诱导成牙本质样细胞在体内外的矿化研究.牙体牙髓牙周病学杂志,2003,13:423-425.
10 Unda FJ, Martín A, Hernandez C, Pérez-Nanclares G, Hilario E, Aréchaga J. FGFs-1 and -2, and TGF beta 1 as inductive signals modulating in vitro odontoblast differentiation. Adv Dent Res. 2001; 15:34-7.
11 于世风.口腔组织病理学.第 4 版.北京:人民卫生出版社,2000.84-85.
12 Arana-Chavez VE, Katchburian E. Freeze-fracture studies of the distal plasma membrane of rat odontoblasts during their differentiation and polarisation. Eur J Oral Sci. 1998; 106(Suppl1): 132-6.
13 Sasagawa I. Mineralization patterns in elasmobranch fish. Microsc Res Tech. 2002; 59(5): 396-407.
14 Linde A. Dentin mineralization and the role of odontoblasts in calcium transport. Connect Tissue Res. 1995; 33(1-3): 163-70.
15 Butler WT. Dentin matrix proteins. Eur J Oral Sci. 1998; 106 (Suppl1): 204-10.
16 Boskey A, Spevak L, Tan M, Doty SB, Butler WT. Dentin sialoprotein (DSP) has limited effects on in vitro apatite formation and growth. Calcif Tissue Int. 2000; 67(6): 472-8.
17 Narayanan K, Srinivas R, Ramachandran A, Hao J, Quinn B, George A. Differentiation of embryonic mesenchymal cells to odontoblast-like cells by overexpression of dentin matrix protein 1. Proc Natl Acad Sci U S A. 2001; 98(8): 4516-21.
18 Thyagarajan T, Sreenath T, Cho A, Wright JT, Kulkarni AB. Reduced expression of dentin sialophosphoprotein is associated with dysplastic dentin in mice overexpressing transforming growth factor-beta 1 in teeth. J Biol Chem. 2001; 276(14):11016-20.
19 Goldberg M, Smith AJ. Cells and extracellular matrices of dentin and pulp: a biological basis for repair and tissue engineering. Crit Rev Oral Biol Med. 2004; 15(1): 13-27.
20 Chai Y, Slavkin HC. Prospects for tooth regeneration in the 21st century: a perspective. Microsc Res Tech. 2003; 60(5):469-79.
21 Ayanoglou CM, Godeau G, Lesty C, Septier D, Goldberg M. CyclosporinA-induced alterations of dentinogenesis in rat molars. J Oral Pathol Med. 1997; 26(3): 129-34.
22 Pekkala E, V?likangas L, Puukka M, Tj?derhane L, Larmas M. The effect of a high-sucrose diet on dentin formation and dental caries in hyperinsulinemic rats. J Dent Res. 2002; 81(8): 536-40.
23 Leonora J, Tj?derhane L, Tieche JM. Parotid gland function and dentin apposition in rat molars. J Dent Res. 2002; 81(4): 259-64.
24 Rowe PS, de Zoysa PA, Dong R, Wang HR, White KE, Econs MJ, Oudet CL. MEPE, a new gene expressed in bone marrow and tumors causing osteomalacia. Genomics.2000;67(1):54
25 Petersen DN, Tkalcevic GT, Mansolf AL, Rivera-Gonzalez R, Brown TA. Identification of osteoblast/osteocyte factor 45 (OF45), a bone-specific cDNA encoding an RGD-containing protein that is highly expressed in osteoblasts and osteocytes. J Biol Chem. 2000;275(46):36172
26 Argiro L, Desbarats M, Glorieux FH, Ecarot B. Mepe, the gene encoding a tumor-secreted protein in oncogenic hypophosphatemic osteomalacia, is expressed in bone. Genomics. 2001;74(3):342.
27 Bresler D, Bruder J, Mohnike K, Fraser WD, Rowe PS. Serum MEPE- ASARM-peptides are elevated in X-linked rickets (HYP): implications for phosphaturia and rickets. J Endocrinol. 2004;183(3):R1
28 Rowe PS, Garrett IR, Schwarz PM, Carnes DL, Lafer EM, Mundy GR, Gutierrez GE. Surface plasmon resonance (SPR) confirms that MEPE binds to PHEX via the MEPE-ASARM motif: a model for impaired mineralization in X-linked rickets (HYP). Bone. 2005; 36(1):33-46.
29 Hayashibara T, Hiraga T, Yi B, Nomizu M, Kumagai Y, Nishimura R, Yoneda T. A synthetic peptide fragment of human MEPE stimulates new boneformation in vitro and in vivo. J Bone Miner Res. 2004; 19(3):455-62.
30 Liu H, Li W, Gao C, Kumagai Y, Blacher RW, DenBesten PK. Dentonin, a fragment of MEPE, enhanced dental pulp stem cell proliferation. J Dent Res. 2004;83(6):496
31 Gowen LC, Petersen DN, Mansolf AL, Qi H, Stock JL, Tkalcevic GT, Simmons HA, Crawford DT, Chidsey-Frink KL, Ke HZ, McNeish JD, Brown TA. Targeted disruption of the osteoblast/osteocyte factor 45 gene (OF45) results in increased bone formation and bone mass. J Biol Chem. 2003;278(3):1998-2007
32 Liu S, Brown TA, Zhou J, Xiao ZS, Awad H, Guilak F, Quarles LD. Role of matrix extracellular phosphoglycoprotein in the pathogenesis of X-linked hypophosphatemia. J Am Soc Nephrol. 2005;16(6):1645-53.
33 Qin C, Baba O, Butler WT. Post-translational modifications of sibling proteins and their roles in osteogenesis and dentinogenesis. Crit Rev Oral Biol Med. 2004;15(3):126
34 Fisher LW, Fedarko NS. Six genes expressed in bones and teeth encode the current members of the SIBLING family of proteins. Connect Tissue Res. 2003; 44 (Suppl 1):33-40.
35 White KE, Lorenz B, Evans WE, Meitinger T, Strom TM, Econs MJ. Molecular cloning of a novel human UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferase, GalNAc-T8, and analysis as a candidate autosomal dominant hypophosphatemic rickets (ADHR) gene. Gene. 2000; 246(1-2): 347-56.
36 Quarles LD, Drezner MK. Pathophysiology of X-linked hypophosphatemia, tumor-induced osteomalacia, and autosomal dominant hypophosphatemia: a perPHEXing problem. J Clin Endocrinol Metab. 2005; 86(2): 494-6.37 Shimada T, Mizutani S, Muto T, Yoneya T, Hino R, Takeda S, Takeuchi Y, Fujita T, Fukumoto S, Yamashita T. Cloning and characterization of FGF23 as a causative factor of tumor-induced osteomalacia. Proc Natl Acad Sci U S A. 2001; 98(11): 6500-5.
38 Murer H, Forster I, Hernando N, Lambert G, Traebert M, Biber J. Posttranscriptional regulation of the proximal tubule NaPi-II transporter in response to PTH and dietary P(i). Am J Physiol. 1999; 277(5 Pt 2): F676-84.
39 Bacic D, Wagner CA, Hernando N, Kaissling B, Biber J, Murer H. Novel aspects in regulated expression of the renal type IIa Na/Pi-cotransporter. Kidney Int Suppl. 2007; (91):S5-S12.
40 Siggelkow H, Schmidt E, Hennies B, Hüfner M. Evidence of downregulation of matrix extracellular phosphoglycoprotein during terminal differentiation in human osteoblasts. Bone. 2004; 35(2):570-6.
41 Liu H, Li W, Shi S, Habelitz S, Gao C, Denbesten P. MEPE is downregulated as dental pulp stem cells differentiate. Arch Oral Biol. 2005; 50(11):923-8.
42 Igarashi M, Kamiya N, Ito K, Takagi M. In situ localization and in vitro expression of osteoblast/osteocyte factor 45 mRNA during bone cell differentiation. Histochem J. 2002; 34(5): 255-63.
43 Nampei A, Hashimoto J, Hayashida K, Tsuboi H, Shi K, Tsuji I, Miyashita H, Yamada T, Matsukawa N, Matsumoto M, Morimoto S, Ogihara T, Ochi T, Yoshikawa H. Matrix extracellular phosphoglycoprotein (MEPE) is highly expressed in osteocytes in human bone. J Bone Miner Metab. 2004; 22(3):176-84.
44 Guo R, Rowe PS, Liu S, Simpson LG, Xiao ZS, Quarles LD. Inhibition of MEPE cleavage by Phex. Biochem Biophys Res Commun. 2002; 297(1):38-45.
45 Liu S, Guo R, Simpson LG, Xiao ZS, Burnham CE, Quarles LD. Regulation of fibroblastic growth factor 23 expression but not degradation by PHEX. J Biol Chem. 2006; 278(39): 37419-26.
46 Quarles LD. Evidence for a bone-kidney axis regulating phosphate homeostasis. J Clin Invest. 2003; 112(5): 642-6.
47 Lu C, Huang S, Miclau T, Helms JA, Colnot C. Mepe is expressed during skeletal development and regeneration. Histochem Cell Biol. 2004; 121(6): 493-9.
48 Jain A, Fedarko NS, Collins MT, Gelman R, Ankrom MA, Tayback M, Fisher LW. Serum levels of matrix extracellular phosphoglycoprotein (MEPE) in normal humans correlate with serum phosphorus, parathyroid hormone and bone mineral density. J Clin Endocrinol Metab. 2004; 89(8): 4158-61.
49 Nagel DE, Khosla S, Sanyal A, Rosen DM, Kumagai Y, Riggs BL. A fragment of the hypophosphatemic factor, MEPE, requires inducible cyclooxygenase-2 to exert potent anabolic effects on normal human marrow osteoblast precursors. J Cell Biochem. 2004; 93(6): 1107-14.
50 胡宝成,薛恒钢,刘爽. 一种新的抗辐射性相关蛋白 AA12 和 CHK1 相互作用的研究.军事医学科学院院刊,2003; 27(5): 345-348.
51 Smits VA, Medema RH. Checking out the G(2)/M transition. Biochim Biophys Acta. 2001; 1519(1-2): 1-12.
52 Khanna KK. Cancer risk and the ATM gene: a continuing debate. J Natl Cancer Inst. 2005; 92(10):795-802.
53 Zoe A, Stewart L, Jennifer A. Cell cycle checkpoints as therapeu tic targets. J Mammary Gland Biol Neoplasia,1999; 4(4): 389-400.
54 Martinez J, Georgoff I, Martinez J, Levine AJ. Cellular localization and cell cycle regulation by a temperature-sensitive p53 protein. Genes Dev. 1991; 5(2):151-9.
55 Hu B, Zhou XY, Wang X, Zeng ZC, Iliakis G, Wang Y. The radioresistance to killing of A1-5 cells derives from activation of the Chk1 pathway. J Biol Chem. 2001; 276(21): 17693-8.
56 胡宝成, 王翔, Iliakis G. 转化的大鼠胚胎成纤维细胞系差异表达基因的筛选研究. 生物技术通讯. 2002;13(2): 118-125.
57 Holm IA, Nelson AE, Robinson BG, Mason RS, Marsh DJ, Cowell CT, Carpenter TO. Mutational analysis and genotype-phenotype correlation of the PHEX gene in X-linked hypophosphatemic rickets. J Clin Endocrinol Metab. 2001; 86(8): 3889-99.
58 Sabbagh Y, Boileau G, DesGroseillers L, Tenenhouse HS. Disease-causing missense mutations in the PHEX gene interfere with membrane targeting of the recombinant protein. Hum Mol Genet. 2005; 10(15): 1539-46.
59 MacDougall M, Simmons D, Gu TT, Dong J. MEPE/OF45, a new dentin/bone matrix protein and candidate gene for dentin diseases mapping to chromosome 4q21. Connect Tissue Res. 2002; 43(2-3): 320-30.
60 Beattie ML, Kim JW, Gong SG, Murdoch-Kinch CA, Simmer JP, Hu JC. Phenotypic variation in dentinogenesis imperfecta/dentin dysplasia linked to 4q21. J Dent Res. 2006; 85(4): 329-33.
61 金冬雁,黎孟枫,译.分子克隆实验指南.第二版.北京:科学出版社.1996
62 颜子颖,王海林,译.精编分子生物学实验指南.北京:科学出版社.1998
63 司徒镇强,吴军正.细胞培养.西安:世界图书出版社西安公司.1996;186
64 Quarles LD, Yohay DA, Lever LW, et al. Distinct proliferative and differentiated stages of murine MC3T3-E1 cells in culture: an in vitro model of osteoblast development. J Bone Miner Res. 1992; 7(6): 683-87
65 Pavlin D, Dove SB, Zadro R, et al. Mechanical loading stimulatesdifferentiation of periodontal osteoblasts in a mouse osteoinduction model: effect on type I collagen and alkaline phosphatase genes. Calcif Tissue Int. 2000; 67(2):163-72.
66 吴奇光.口腔组织病理学.北京:人民卫生出版社.1993;70
67 Robey PG. Bone matrix proteoglycans and glycoprotein. Principles of Bone Biology. Academic Press, San Diego,Ca,USA, 1996;155
68 Horiuch K, Amizuka N, Takeshita S. Identification and Characterization of a noval protein, Periostin, with restricted expression to periosteum and periodontal ligament and increased expression by transforming growth factor β. J Bone Miner Res. 1999;14:1239
69 Hunter GK, Goldberg HA. Nucleation of hydroxyapatite by bone sialoprotein. Proc Natl Acad Sci U S A,1993;90:8562
70 颜子颖,王海林,译.精编分子生物学实验指南.北京:科学出版社.1998
71 Kaufman RJ. Overview of Vectors used for expression in mammalian cells. Methods enzymol. 1990; 185: 487-527
72 Sampath TK, Maliakal JC, Hauschka PV et al. Recombinant human osteogenic protein-1 (hOP-1) induces new bone formation in vivo with a specific activity comparable with natural bovine osteogenic protein and stimulates osteoblast proliferation and differentiation in vitro. J Biol Chem 1992 ;267(28):20352-62