摘要
骨和牙本质细胞外基质(ECM)均含有胶原和非胶原蛋白(noncollagenous proteins,NCPs)。小整合素结合配体N连接的糖蛋白(small integrin-binding ligand,n-linked glycoprotein family,SIBLING),是NCPS的一类。其成员包括:牙本质基质蛋白1(dentin matrix protein 1,DMP 1)、牙本质涎磷蛋白(dentin sialophosphoprotein,DSPP)、骨涎蛋白(bone sialoprotein, BSP)、骨桥蛋白(osteopontin, OPN)、基质细胞外磷酸糖蛋白(matrix extracellular phosphoglycoprotein,MEPE)。这五种蛋白的生物学特性主要是在骨组织形成和牙齿发育过程中起着重要的发育调控作用。DMP 1在蛋白水解酶的作用下水解为两个具有活性的功能片段:57kDa (C-DMP1)和37kDa (N-DMP1)。C-DMP1和N-DMP1蛋白片段不仅组织的属性和蛋白结构不同,而且在细胞和组织中分布部位也不相同, C-DMP1蛋白主要在矿化的牙本质中存在表达,而N-DMP1蛋白表达主要位于非矿化的前期牙本质。因此提示C-DMP1和N-DMP1蛋白可能具有不同的生物学作用。牙本质涎磷蛋白(DSPP),经过类似于DMP1的蛋白水解活化过程后形成牙本质涎蛋白(DSP)和牙本质磷蛋白(DPP)。骨涎蛋白(BSP)是一种磷酸化硫化糖蛋白,在成熟的骨细胞中表达,而未成熟的前体细胞不表达,蛋白特异性分布于矿化组织,是成骨细胞分化的标志。骨桥蛋白(OPN)是一种分泌型磷酸化糖蛋白,体内外研究均表明OPN可以有效抑制羟基磷灰石晶体的合成和生长,在牙骨质和牙本质形成过程中具有抑制和调节矿化的作用。
一直以来,学者对于SIBLING蛋白的研究主要集中在大鼠等动物的牙齿发育及功能,而对于SIBLING蛋白在人牙及牙周组织的分布情况和意义尚无研究报道。因此,我们设计了本实验,利用SIBLING蛋白家族成员中抗C-DMP1、N-DMP1、DSP、BSP、OPN蛋白的五种特异性抗体,通过免疫组化技术,研究C-DMP1、N-DMP1、DSP、BSP、OPN五种蛋白在人牙及牙周组织中的表达情况,并探讨他们的表达意义。本实验对象选取30例吉林大学口腔医学院颌面外科颌骨恶性肿瘤切除的牙及牙周组织标本,制作牙及牙周组织联合切片。采用PV二步免疫组化的方法进行上述五种抗体的免疫组织化学染色。染色后观察C-DMP1、N-DMP1、DSP、BSP、OPN蛋白的五种抗体在人牙及牙周组织中的表达部位及探讨这五种抗体分布的意义。
对实验结果讨论分析后得到以下的结论:一、DMP1蛋白的不同活化形式(N-DMP1和C-DMP1)在人牙及牙周组织中分布位置不同,进一步证实了N-DMP1和C-DMP1在组织矿化过程中具有不同的生物学特性;二、DMP1和DSPP在人牙齿发育过程和形成维持过程中起到协同和互补的作用。三BSP、OPN在人牙及牙周组织中不同部位表达程度不同,可能均参与基质的矿化,并具有不同的作用。
The extracellular matrix (ECM) of bone and dentin contains collagen and noncollagenous proteins (NCPs). The Small Integrin-Binding LIgand, N-linked Glycoprotein (SIBLING) family is one category of NCPs, which includes dentin matrix protein 1 (DMP1), dentin sialophosphoprotein (DSPP), bone sialoprotein (BSP) ,osteopontin (OPN) and MEPE. The SIBLING family members are expressed in the mineralized tissues of bone and dentin and they believed to play important roles in the mineralization.
In the ECM of bone and dentin, DMP1 mainly occurs as the proteolytically processed 37 kDa (DMP1 N-terminal) and 57 kDa (DMP1 C-terminal) fragments, which originate from the N-terminal and C-terminal regions of the DMP1 amino acid sequence respectively. The distribution of DMP1 N- and DMP1 C-terminal fragments in dentin are different: the DMP1 N-terminal is located in the unmineralized predentin while the DMP1 C-terminal is mainly found in the mineralized dentin, which suggest that these two fragments may play different roles in dentinogenesis. DSPP in the dentin and bone is present as the proteolytically processed fragments: dentin sialoprotein (DSP) and dentin phosphoprotein (DPP). BSP is primarily found in bone, cementum and tertiary dentin. The biological functions of BSP in the mineralized tissues are complex. Studies showed that BSP acts as a nucleator for the formation of initial hydroxyapatite(HA) crystals, and then, as this mineral grows on the collagen matrix, it acts as an inhibitor in directing the growth of the crystals. In the mineralized tissues, OPN is mainly found in the bone, cementum, predentin, and tertiary dentin. Both in vitro and in vivo studies have shown that OPN is an effective inhibitor of HA formation and growth.
The experiment objects are 30 human teeth and affiliated periodontal tissues specimens,. The expression of SIBLING proteins which contains C-DMP1、N-DMP1、DSP、BSP、OPN were examined in human teeth and affiliated periodontal tissues by immunohistochemical technique. Immunohistochemical staining procedure was performed according to PV method.
In the result,we find that :
1)C-DMP1 were mainly expressed in peritubular dentin, dentinal tubules ,odontoblast and cementum, while were negtively expressed in PDL tissue and predentin.
2 ) N-DMP1 were positively expressed in predentine, odontoblast cell,and strong positively expressed in PDL cell and periodontal ligament which were negtively expressed in cementum.
3)OPN were positively expressed in predentine, odontoblast, alveolar bone, cementum and periodontal ligament which were negtively expressed in dentin.
4)BSP were positively expressed in periodontal ligament,and strongly positively expressed in cementum, osteoblast, osteoid and alveolar bone, and which were negtively expressed in predentine and dentin.
5 ) DSP were mainly expressed in dentinal tubules,and weekly expressed in odontoblast.There was no expression of DSP in predentine, alveolar bone, cementum, periodontal ligament and gingiva.
According to the observation of the results above all, we can conclude that:
1.The distribution of DMP1 N- and DMP1 C-terminal fragments in dentin are different further comfirm their different functions.
2.DMP1 and DSP play complementary and/or synergistic roles in the formation of human healthy teeth.
3.BSP and OPN have different expression levels at different locations in human teeth and periodontal tissues, they may different play important roles in the mineralization of bone or dentin matrix..
引文
[1]Fisher L W, Torchia DA, Fohr B, et al. Biochem Biophys Res Commun,2001,280(2):460-465.
[2]Fisher LW, Fedarko NS. Connect Tiss Res,2003,44(Suppl 1):33-40.
[3]Hu CC, Hart TC, Dupont BR, et al. J Dent Res,2000, 79(4):912-919.
[4]Chen J, Sasaguri K, Sodek J, et al. Europ J Oral Sci,1998,106(Suppl 1):331-336.
[5]Rowe PS, de Zoysa P, Dong R, et al.Genomics,2000,67(1):54-68.
[6]MacDougall M, DuPont BR, Simmons D, et al. Cytogenet Cell Genet,1996,74(3):189.
[7]Hirst KL, Ibaraki-O’Connor K, Young MF, et al.J DenRes,1997,76(3):754-760.
[8]Feng JQ, Huang H, Lu Y, et al.J Dent Res,2003,82(10):776-780.
[9]Dsouza.RN,Bronekers.AL,HapPonen.RP. Developmental expression of a 53kD dentin sialoProtein in rat tooth organs. J Histochem Cytochem.1992:40(3):359-366
[10]Qin C, Brunn J。C, Cadena E, et al.Connect Tiss Res,2003,44(Suppl 1):179-183.
[11]Sodek J, Ganss B, McKee MD. Crit Rev Oral Biol Med,2000,11(3):279-303.
[12]Jain A, Karadag A, Fohr B, et al. J Biol Chem,2002, 277(16):13700-13708.
[13]顾天爵,冯宗忱,于秉治,等.生物化学.北京:科学出版社,1995:277-278.
[14]Qin C,Baba O, Butler WT. Crit Rev Oral Biol Med,2004,15(3):126-136.
[15]Ogbureke KUE, Fish LW.J Dent Res,2004,83(9):664-670.
[16]Fedarko NS, Jain A, Karadag A, et al. FASEB J,2004,18(6):734-736.
[17]Jain A, Karadag A, Fohr B, et al. J Biol Chem,2002, 277(16):13700-13708.
[18]Giachelli CM, Steitz S. Matrix Biol, 2000,19(7):615-622.
[19]Fedarko NS, Fohr B, Robey PG, et al. J Biol Chem,2000,275(22):16666-16672.
[20]Hirst KL, Ibaraki-O′Connor K, Young MF, et al. J DentRes,1997,76(3):754-760.
[21]Toyosawa S, Tomita Y, Kishino M, et al. Mod Pathol,2004,17(5):573-578.
[22]Narayanan K, Srinivas R, Ramachandran A, et al. Proc Natl Acad Sci U S A,2001,98(8):4516-4521.
[23]Almushayt A, Narayanan K, Zaki AE, et al. Gene Ther,2006,13(7):611-620.
[24]Lu Y, Ye L, Yu SB, et al. Dev Biol,2007,303(1):191-201.
[25]He G,Dahl T, Veis A, et al. Connect Tissue Res,2003,44(Suppl 1):240-245.
[26]He G, George A.J Biol Chem,2004,279(12):11649-11656.
[27]He G, Gajeraman S, Schultz D, et al.Biochemistry,2005,44(49):16140-16148.
[28]Feng JQ, Ward LM, Liu S, et al. Nat Genet,2006,38(11):1310-1315.
[29]Terasawa M, Shimokawa R, Terashima T, et al. J BoneMiner Metab,2004,22(5):430-438.
[30]I.R.Dickson, M.T. Dimuzio, D.Volpin, S. Ananthanarayanan, and A. Veis, "The extraction of phosphoproteins from bovine dentin, "Calcif Tissue Res,vol.19,pp.51-61,Nov 24 1975.
[31]M.T.Dimuzio and A. Veis, "Phosphophoryns-major noncollagenous proteins of rat incisor dentin," CalcifTissue Res,vol.25,pp.169-78,May 26 1978.
[32]M. MacDougall, D. Simmons, X. Luan, J. Nydegger, J.Feng,and T. T.Gu,"Dentin phosphoprotein anddentin sialoprotein are cleavage products expressed from a single transcript coded by a gene on humanchromosome 4.Dentin phosphoprotein DNA sequence determination, "J Biol Chem,vol.272,pp.835-42,Jan 10 1997.
[33]L.W.Fisher,A.Jain,M.Tayback,and N.S.Fedarko,"Small integrin binding ligand N-linked glycoprotein gene family expression in different cancers,"Clin Cancer Res,vol.10,pp.8501-11,Dec 15 2004.
[34]M.Chaplet, D.Waltregny, C. Detry, L.W.Fisher, V.Castronovo, and A.Bellahcene,"Expression of dentin sialophosphoprotein in human prostate cancer and its correlation with tumor aggressiveness, "Int J Cancer,vol.118,pp.850-6,Feb 15 2006.
[35]A.George,L.Bannon,B.Sabsay,J.W.Dillon,J.Malone,A.Veis,N.A.Jenkins,D.J.Gilbert,and N.G.Copeland," The carboxyl-terminal domain of phosphophoryn contains unique extended triplet amino acidrepeat sequences forming ordered carboxyl-phosphate interaction ridges that may be essential in the biomineralization process,"J Biol Chem,vol.271,pp.32869-73,Dec 20 1996.
[36]Y. Nakamura, I. Slaby,K.Matsumoto,H.H.Ritchie,and S.P.Lyngstadaas,"Immunohistochemical characterization of rapid dentin formation induced by enamel matrix derivative,"Calcif Tissue Int,vol.75,pp.243-52,Sep 2004.
[37]J.A.Jadlowiec,X.Zhang,J.Li,P.G.Campbell,and C.Sfeir,"Extracellular matrix-mediated signaling bydentin phosphophoryn involves activation of the Smad pathway independent of bone morphogeneticprotein,"J Biol Chem,vol.281,pp.5341-7,Mar 3 2006.
[38]S.Chen,L.Chen,A.Jahangiri,B.Chen,Y.Wu,H.H.Chuang,C.Qin,andM.MacDougall,"Expressionand processing of small integrin-binding ligand N-linked glycoproteins in mouseodontoblastic cells,"ArchOral Biol,vol.53,pp.879-89,Sep 2008.
[39]T.Sreenath,T.Thyagarajan,B.Hall,G.Longenecker,R.D'Souza,S.Hong,J.T.Wright,M.MacDougall,J.Sauk,and A.B.Kulkarni,"Dentin sialophosphoprotein knockout mouse teeth display widened predentinzone and develop defective dentin mineralization similar to human dentinogenesis imperfecta type III,"J Biol Chem,vol.278,pp.24874-80,Jul 4 2003.
[40]Bellahcene A, Antoine N, Clausse N, et al. Lab Inv,1996,75(2):203-210.
[41]Chenu C, Delmas P.J Bone Miner Res,1992,7:45-47.
[42]Chen J, Shapiro HS, Sodck J.J Bone Miner Res, 1992,8:987-997.
[43]MacNeil RL,Berry J,Strayhorn C,et al.Arch Oral Biol,1996,41:827-835.
[44]Sasaguri K, Ganss B, Sodek J, et al. J Arch Oral Biol,2000,45(7):551-553.
[45]D′Errico JA, Macneil RL, Takata J, et al.Bone,1997,26(1):10-16.
[46]Pituru S, McCulloch CAG, Narayanan AS, et al.J Pe-riodont Res,1994,29(2):81-94.
[47]Ong Chunming, Goldschmidt-Clermont, Pascal J. Circul-ation Res,2000,86(8):827-828.
[48]Goldsmith HL,Labrosse JM, Maintosh FA et al. Homotypic interactions of soluble and immobilized osteopontin. ANN Biomed Eng, 2002; 30 (6): 840-850.
[49]Denhardt DT, Guo XJ. Osteopontin: a protein with diverse functions[J]. FASEB, 1993,7(12):1475.
[50]Reinholt FP, Hultenby K, OldbergA, et al. Osteopontin--a possible an-chor of osteoclasts to bone[J]. Proc Natl Acad Sci USA, 1990,87(12):4473.
[51]Mark MP, Prince CW, Oosawa T, et al. Inmunohistochemical demonstration of a 44-kD phosphoprotein in developing rat bones[J]. Histochem Cytochem, 1987,35(7):707.
[52]王立平,党耕町.骨桥蛋白在骨愈合中的基因表达[J].北京医科大学学报,1998,30(4):355.
[53]Oldberg A, Franzen A, Heinegard D. Cloning and sequence analysis of rat bone sialoprotein(osteopontin) cDNA reveals an Arg-Gly-Aspcell-binding sequence[J]. Prog Natl Acad Sci USA, 1986,83:8819.
[54]Somerman MJ, Prince CW, Sauk JJ, et al. Mechanism of fibroblast attachment to bone extracellular matrix:role of a 44 kilodelton bone phosphoprotein[J]. Bone Miner Res, 1987,2(3):259.
[55]Hunter GK, Goldberg HA. Modulation of crystal formation by bone phosphoproteins:role of glutamic acidrich sequences In the nucleation of hydroxyapatite bybone sialoprotein[J]. Biochem, 1994,302(Pt 1):175.
[56]张红军.骨桥蛋白的功能及与消化系统等疾病的关系[J].国外医学·生理病理及临床分册,1999,19(6):524.
[57]高建军.破骨细胞成熟和活化以及骨吸收机制[J].国外医学·内分泌分册,1998,18(2):57.
[58]刘大为,傅民魁,李盛琳,等.机械牵张作用对细胞骨桥蛋白和表达的影响[J].中国口腔医学杂志,2000,35(1):27.
[59]王渝,臧光祥,孙宏晨等.提高牙及牙周组织联合切片石蜡质量的探讨.中华口腔医学杂志,2005;40(4):437-438.
[60]Lorenz-Depiereux B, Bastepe M, Benet-Pages A,et al.DMP1 mutations in autosomal recessive hypophosphatemia implicate a bonematrix protein in the regulation ofphosphate homeostasis[J].NatGenet, 2006, 3(8): 1248-1250.
[61]TerasawaM, Shimokawa R, Terashima T,et al. Expression of dentinmatrix protein 1 (DMP1) in nonmineralized tissues[J]. JBoneMinerMetab, 2004, 22(5): 430-438.
[62]OgburekeKU, FisherLW. SIBLING expression patterns in ductepithelia reflect the degree ofmetabolic activity[J].Histochem Cytochem, 2007, 55(4): 403-409.
[63]逄键梁,吴补领,张亚庆,等.人牙髓干细胞诱导后人牙本质基质蛋白1基因转录活性的变化和意义[J].牙体牙髓牙周病学杂志, 2007, 17(9): 494-498.
[64]Huang B, Maciejewska I, Sun Y,et al. Identification of fulllength dentin matrix protein 1 in dentin and bone[ J].Calcif Tissue Int,2008, 82(5): 401-410.
[65]O.Baba,C.Qin,J.C.Brunn,J.N.Wygant,B.W.McIntyre,and W.T.Butler,"Colocalization of dentinmatrix protein 1 and dentin sialoprotein at late stages of rat molar development,"Matrix Biol,vol.23,pp.371-9,Oct 2004.
[66]C.Qin,J.C.Brunn,R.G.Cook,R.S.Orkiszewski,J.P.Malone,A.Veis,and W.T.Butler,"Evidence forthe proteolytic processing of dentin matrix protein 1.Identification and characterization of processed fragments and cleavage sites,"J Biol Chem, vol.278, pp.34700-8,Sep 5 2003.
[67]K.Narayanan,S.Gajjeraman,A.Ramachandran,J.Hao,and A.George,"Dentin matrix protein 1 regulates dentin sialophosphoprotein gene transcription during early odontoblast differentiation,"J Biol Chem,vol.281,pp.19064-71,Jul 14 2006.
[68]Bronckers AL , Farach2Cars on MC , Van Waveren E , et al1Immunolocalization of osteopontin , osteocalcin , and dentin sialoprotein during dental root formation and earlycementogenesis in the rat1 J Bone Miner Res , 1994 ,9 :83328411
[69]Bellahcene A,Antoine N,Clausse N,et al.Lab Inv,1996,75(2):203-210.
[70]刘宏,王勤涛,吴织芬,等.rhBMP-2对人牙牙周细胞骨桥蛋白表达的影响[J].中华口腔医学杂志,2000,35(5):330.
[71]Denhartdt DT, Guo X. Osteopontin:a protein with diverse functions[J].FASEB, 1993,7(15):1475.
[72]D.Septier,M.A.Torres-Quintana,S.Menashi,A.George,andM.Goldberg,"Inositolhexasulphate,acasein kinase inhibitor,alters the distribution of dentin matrix protein 1 in cultured embryonic mouse toothgerms,"Eur J Oral Sci,vol.109,pp.198-203,Jun 2001.
[73]C.Qin,J.C.Brunn,J.Jones,A.George,A.Ramachandran,J.P.Gorski,andW.T.Butler,"Acomparativestudy of sialic acid-rich proteins in rat bone and dentin,"Eur J Oral Sci,vol.109, pp.133-41,Apr 2001.
[74]Reinholt FP, Hultenby K, OldbergA, et al. Osteopontin--a possible anchor of osteoclasts to bone[J]. Proc Natl Acad Sci USA, 1990,87(12):4473.
[75]Qin C, Brunn J.C, Cadena E, et al. J Dent Res,2002, 81(6):392-394.