人牙本质涎蛋白的基因克隆、表达和功能研究
|
摘要
牙本质涎蛋白(dentin sialoprotein，DSP)属于牙本质非胶原蛋白，占牙本质非胶原蛋白的5％～8％，因与骨涎蛋白(bone sialoprotein，BSP)相似而得名。DSP富含谷氨酸(Glu)、天冬氨酸(Asp)、丝氨酸(Ser)和甘氨酸(Gly)，由366个氨基酸组成，分子量约为53 000，含30％的碳水化合物和10％的涎酸，有6个潜在的糖基化位点和13个潜在的磷酸化位点。DSP的功能尚不清楚，考虑到DSP在成牙本质细胞中有阳性表达，并在前成釉细胞中有一过性表达，推测DSP可作为成牙本质细胞和相关细胞的标记物，并可能在上皮和间充质之间充当信号分子，参与成牙本质细胞分化。
一、人牙本质涎蛋白的基因克隆、表达、多抗制备和组织表达研究
克隆hDSP成熟肽编码区基因片段并进行原核表达，用异硫氰酸胍一步法从人牙乳头组织中抽提总RNA，再用Oligo(dt)作引物逆转录合成牙乳头cDNA，然后用RT-PCR法，从cDNA中扩增出hDSP基因片段(约600bp)，将所得基因片段插入pBluescript质粒载体，转化到大肠杆菌XL1-Blue后，挑选阳性克隆，提取重组质粒DNA，通过酶切分析和核苷酸序列分析鉴定阳性克隆；再将hDSP基因重组入表达载体pGEX-3X中构建表达载体，并在大肠杆菌BL21中进行表达。结果酶切图谱和序列分析与文献报道一致，并且hDSP蛋白(约200个氨基酸)在大肠杆菌中得到了高效表达。用原核表达并经过纯化
第囚旱巨大学傅士学位论文
的人牙本质涎蛋白免疫新西兰大白兔获得多克隆抗血清，抗血清效价可达1：
100 000。用 Western blot法检测不同组织的表达情况，结果显示 hDSP在人牙
胚中有表达，分子量约为60 000左右，而其它软组织（包括牙龈、肝、肾。
脾等）则无表达，其分子量大小与天然牙本质中DSP的分子量接近。
二、人牙本质涎蛋白全长基因表达载体的构建和莫核细胞转染
用原核系统表达目的蛋白具有产量高、成本低、操作简单等特点，但是原
核系统表达的目的蛋白不具备折叠、糖基化等特点，所以用原核系统表达的目
的蛋白常常不具备天然蛋白的功能。由于hDSP是高度糖基化的糖蛋白，所以，
最好选择真核表达系统。本研究将hDSP全长基因克隆入真核表达载体
pCDNA3中，构建pCDNA3－hDSP重组质粒。酶切鉴定和测序正确后，将
pcDNA3－hDSP真核表达质粒转染COSJ细胞，Western Blot和免疫组化均证
明hDSP在COS刁细胞中得到了较高表达，为其功能研究提供了保障。
三、人牙本质涎蛋白的功能研究
首先，通过免疫组化方法观察了hDSP在人牙胚不同发育阶段的表达情
况。显示hDSP在蕾状期、帽状期成釉器上皮、钟状早期内釉上皮有弱表达，
正在分泌基质的成牙本质细胞、牙本质小管有强阳性表达，而在钟状晚期，
hDSP主要在牙本质小管中表达，成牙本质细胞转为弱阳性表达。前成釉细胞、
成釉细胞有一过性表达，而前期牙本质始终无阳性表达，牙胚周围牙槽骨、软
骨和口腔软组织无阳性表达，提示hDSP是牙齿特异性的蛋白，它只在成牙组
织中有表达。另外hDSP在前成釉细胞和成釉细胞中的一过性表达，提示hDSP
不仅参与了牙本质的形成，而且还可能参与了上皮和间质之间的信号传递。再
者前期牙本质阴性表达，表明hDSP蛋白直接通过成牙本质细胞突起穿过前期
牙本质分泌至矿化前沿，参与牙本质的形成。
其次，观察了hDSP对体外培养的人牙乳头间充质细胞增殖和碱性磷酸酶
活性的影响。选择第5代人牙乳头间充质细胞，通过MTT比色法观察细胞增
殖情况，发现hD盯能明显抑制培养的人牙乳头间充质细胞增殖（P＜0刀1）；通
4
第q罩巨大学俗士学位论X
过检测细胞内和细胞外碱性磷酸酶活性，发现hDSP能明显促进培养的人牙乳
头间充质细胞碱性磷酸酶的分泌（P＜o．01），提示hD SP在人牙乳头间充质细
胞的分化和矿化过程中可能起重要的调节作用。
最后，通过hDSP对狗牙直接盖髓观察hDSP对牙髓损伤修复的影响。选
择体重 10 kg左右、年龄 6～12月的健康杂种狗 3只，选用尖牙、磨牙为实验
牙，共30个。分实验和对照两组：实验组：hDSP表达上清浓缩液加胶原膜，
18个牙；对照组：空白载体表达上清浓缩液加胶原膜和 PBS加胶原膜，各 6
个牙。胶原膜作为载体与实验组、对照组的液体复合，4”C过夜。
结果：hDSP盖髓术后2周，对照组和实验组牙髓组织炎性细胞少见，成
纤维细胞增生明显，实验组和对照组均无牙本质桥形成。随着盖髓时间的延长
Dentin sialoprotein (DSP), comprising 5%-8% of noncollagenous proteins, is found exclusively in the extracts of dentin extracellular matrix. This protein was so named, because of its overall resemblance to bone sialoprotein (BSP). DSP is rich in aspartic acid (Asp) , serine (Ser) , glutamie acid (Glu) and glycine (Gly) . Sedimentation equilibrium analysis revealed that DSP has a molercular weight of about 53,000 and approximately 366 amino acids. The 30% carbohydrate included about 10% sialic acid. The cDNA sequence analysis indicated that DSP has six potential N-glycosylation sites and thirteen potential phosphorylation sites. While the function of DSP is still unknown, DSP transcripts were found to be expressed first in preameloblasts, then in young odontoblasts, and finally substantially in mature odontoblasts actively synthesizing dentin. This transient expression profile may suggest that DSP is an important marker of the odontoblast and related cells and take part in dentinogenesis by serving as a signal molecule between epithelia-mesenchyme.
1. Cloning, expression, preparation of potyclonal anti-sera and tissue distribution of hDSP
In the present study, total RNA was extracted from the tooth germs of a
legally aborted human embryo by acid guanidinium thiocyanata-phenol-choroform method, the desired DNA product was obtained from the total RNA by RT-PCR with the primers including Oligo(dt) and two gene specific ones. The segment (about 600 bp) was inserted into a pBluescript vector and the interesting plasmid was transformed into E.Coli host strain XL 1-Blue. Reconstructed plasmid of pBS-hDSP was analyzed by restriction endonuclease mapping and DNA sequencing, then hDSP was cloned into expression vector pGEX-3X and expressed in E.coli BL21. The results showed that the restriction endonuclease map and sequence of hDSP encoding mature protein were consistent with those published and hDSP protein could be efficiently expressed in prokaryocytes.
Polyclonal antibodies of hDSP were prepared by immunizing New Zealand rabbit with the purified hDSP obtained by prokaryotic expression. The tilter of the prepared antibody of hDSP was 1: 100 000 at least. The expression of hDSP was observed in human tooth germs and other tissues by Western blot. The results showed that hDSP was positive in tooth germs, but negative hi other human tissues including gingiva, liver, kidney and spleen. Its molecular weight was about 60, 000. The size was similar to that of DSP in native dentin.
2. Construction and transfection of the expression vector of a full length gene of hDSP
Foreign protein expression by prokaryocytes possesses the features of high output, low cost and simple methods, but the expressed protein cannot be folded and glycosylated. Thus, such protein often doesn't have the native protein's functions. Since hDSP is a sort of glycoproteins, we selected mammalian cell as expression system. Recombind plasmid of pcDNA3-hDSP was constructed by inserting full length of hDSP into pcDNAS and transfected into COS-7 cell after identified by enzyme analysis and seqencing. Western Blot and immunochemistry showed that hDSP could be expressed in COS-7. This laid a solid foundation for
further functional study.
3. Functional study of h DSP
The expression of hDSP in human tooth germs was examined by immunochemistry. The result showed that hDSP first appeared weakly in enamel epithelial cells of bud and cap stages, then strongly in functional odontoblasts and dentin tubules of the bell stage. With the development of the tooth germs, resting odontoblasts, cells associated with a full thickness of dentin, appeared weakly reactive with anti-hDSP antibodies. Pre-odontoblasts and pre-dentin were always negative. Pre-ameloblasts and ameloblasts were transiently stained positive. This may suggest that hDSP protein is transported via odontoblastic processes through the pre-dentin and deposited directly at the mineralization front during dentin formation and hDSP is involved in cooperation with signaling molecules in pr
一、人牙本质涎蛋白的基因克隆、表达、多抗制备和组织表达研究
克隆hDSP成熟肽编码区基因片段并进行原核表达，用异硫氰酸胍一步法从人牙乳头组织中抽提总RNA，再用Oligo(dt)作引物逆转录合成牙乳头cDNA，然后用RT-PCR法，从cDNA中扩增出hDSP基因片段(约600bp)，将所得基因片段插入pBluescript质粒载体，转化到大肠杆菌XL1-Blue后，挑选阳性克隆，提取重组质粒DNA，通过酶切分析和核苷酸序列分析鉴定阳性克隆；再将hDSP基因重组入表达载体pGEX-3X中构建表达载体，并在大肠杆菌BL21中进行表达。结果酶切图谱和序列分析与文献报道一致，并且hDSP蛋白(约200个氨基酸)在大肠杆菌中得到了高效表达。用原核表达并经过纯化
第囚旱巨大学傅士学位论文
的人牙本质涎蛋白免疫新西兰大白兔获得多克隆抗血清，抗血清效价可达1：
100 000。用 Western blot法检测不同组织的表达情况，结果显示 hDSP在人牙
胚中有表达，分子量约为60 000左右，而其它软组织（包括牙龈、肝、肾。
脾等）则无表达，其分子量大小与天然牙本质中DSP的分子量接近。
二、人牙本质涎蛋白全长基因表达载体的构建和莫核细胞转染
用原核系统表达目的蛋白具有产量高、成本低、操作简单等特点，但是原
核系统表达的目的蛋白不具备折叠、糖基化等特点，所以用原核系统表达的目
的蛋白常常不具备天然蛋白的功能。由于hDSP是高度糖基化的糖蛋白，所以，
最好选择真核表达系统。本研究将hDSP全长基因克隆入真核表达载体
pCDNA3中，构建pCDNA3－hDSP重组质粒。酶切鉴定和测序正确后，将
pcDNA3－hDSP真核表达质粒转染COSJ细胞，Western Blot和免疫组化均证
明hDSP在COS刁细胞中得到了较高表达，为其功能研究提供了保障。
三、人牙本质涎蛋白的功能研究
首先，通过免疫组化方法观察了hDSP在人牙胚不同发育阶段的表达情
况。显示hDSP在蕾状期、帽状期成釉器上皮、钟状早期内釉上皮有弱表达，
正在分泌基质的成牙本质细胞、牙本质小管有强阳性表达，而在钟状晚期，
hDSP主要在牙本质小管中表达，成牙本质细胞转为弱阳性表达。前成釉细胞、
成釉细胞有一过性表达，而前期牙本质始终无阳性表达，牙胚周围牙槽骨、软
骨和口腔软组织无阳性表达，提示hDSP是牙齿特异性的蛋白，它只在成牙组
织中有表达。另外hDSP在前成釉细胞和成釉细胞中的一过性表达，提示hDSP
不仅参与了牙本质的形成，而且还可能参与了上皮和间质之间的信号传递。再
者前期牙本质阴性表达，表明hDSP蛋白直接通过成牙本质细胞突起穿过前期
牙本质分泌至矿化前沿，参与牙本质的形成。
其次，观察了hDSP对体外培养的人牙乳头间充质细胞增殖和碱性磷酸酶
活性的影响。选择第5代人牙乳头间充质细胞，通过MTT比色法观察细胞增
殖情况，发现hD盯能明显抑制培养的人牙乳头间充质细胞增殖（P＜0刀1）；通
4
第q罩巨大学俗士学位论X
过检测细胞内和细胞外碱性磷酸酶活性，发现hDSP能明显促进培养的人牙乳
头间充质细胞碱性磷酸酶的分泌（P＜o．01），提示hD SP在人牙乳头间充质细
胞的分化和矿化过程中可能起重要的调节作用。
最后，通过hDSP对狗牙直接盖髓观察hDSP对牙髓损伤修复的影响。选
择体重 10 kg左右、年龄 6～12月的健康杂种狗 3只，选用尖牙、磨牙为实验
牙，共30个。分实验和对照两组：实验组：hDSP表达上清浓缩液加胶原膜，
18个牙；对照组：空白载体表达上清浓缩液加胶原膜和 PBS加胶原膜，各 6
个牙。胶原膜作为载体与实验组、对照组的液体复合，4”C过夜。
结果：hDSP盖髓术后2周，对照组和实验组牙髓组织炎性细胞少见，成
纤维细胞增生明显，实验组和对照组均无牙本质桥形成。随着盖髓时间的延长
Dentin sialoprotein (DSP), comprising 5%-8% of noncollagenous proteins, is found exclusively in the extracts of dentin extracellular matrix. This protein was so named, because of its overall resemblance to bone sialoprotein (BSP). DSP is rich in aspartic acid (Asp) , serine (Ser) , glutamie acid (Glu) and glycine (Gly) . Sedimentation equilibrium analysis revealed that DSP has a molercular weight of about 53,000 and approximately 366 amino acids. The 30% carbohydrate included about 10% sialic acid. The cDNA sequence analysis indicated that DSP has six potential N-glycosylation sites and thirteen potential phosphorylation sites. While the function of DSP is still unknown, DSP transcripts were found to be expressed first in preameloblasts, then in young odontoblasts, and finally substantially in mature odontoblasts actively synthesizing dentin. This transient expression profile may suggest that DSP is an important marker of the odontoblast and related cells and take part in dentinogenesis by serving as a signal molecule between epithelia-mesenchyme.
1. Cloning, expression, preparation of potyclonal anti-sera and tissue distribution of hDSP
In the present study, total RNA was extracted from the tooth germs of a
legally aborted human embryo by acid guanidinium thiocyanata-phenol-choroform method, the desired DNA product was obtained from the total RNA by RT-PCR with the primers including Oligo(dt) and two gene specific ones. The segment (about 600 bp) was inserted into a pBluescript vector and the interesting plasmid was transformed into E.Coli host strain XL 1-Blue. Reconstructed plasmid of pBS-hDSP was analyzed by restriction endonuclease mapping and DNA sequencing, then hDSP was cloned into expression vector pGEX-3X and expressed in E.coli BL21. The results showed that the restriction endonuclease map and sequence of hDSP encoding mature protein were consistent with those published and hDSP protein could be efficiently expressed in prokaryocytes.
Polyclonal antibodies of hDSP were prepared by immunizing New Zealand rabbit with the purified hDSP obtained by prokaryotic expression. The tilter of the prepared antibody of hDSP was 1: 100 000 at least. The expression of hDSP was observed in human tooth germs and other tissues by Western blot. The results showed that hDSP was positive in tooth germs, but negative hi other human tissues including gingiva, liver, kidney and spleen. Its molecular weight was about 60, 000. The size was similar to that of DSP in native dentin.
2. Construction and transfection of the expression vector of a full length gene of hDSP
Foreign protein expression by prokaryocytes possesses the features of high output, low cost and simple methods, but the expressed protein cannot be folded and glycosylated. Thus, such protein often doesn't have the native protein's functions. Since hDSP is a sort of glycoproteins, we selected mammalian cell as expression system. Recombind plasmid of pcDNA3-hDSP was constructed by inserting full length of hDSP into pcDNAS and transfected into COS-7 cell after identified by enzyme analysis and seqencing. Western Blot and immunochemistry showed that hDSP could be expressed in COS-7. This laid a solid foundation for
further functional study.
3. Functional study of h DSP
The expression of hDSP in human tooth germs was examined by immunochemistry. The result showed that hDSP first appeared weakly in enamel epithelial cells of bud and cap stages, then strongly in functional odontoblasts and dentin tubules of the bell stage. With the development of the tooth germs, resting odontoblasts, cells associated with a full thickness of dentin, appeared weakly reactive with anti-hDSP antibodies. Pre-odontoblasts and pre-dentin were always negative. Pre-ameloblasts and ameloblasts were transiently stained positive. This may suggest that hDSP protein is transported via odontoblastic processes through the pre-dentin and deposited directly at the mineralization front during dentin formation and hDSP is involved in cooperation with signaling molecules in pr
引文
1． Butler WT. Dentin specific proteins. Methods Enzymol, 1987, 145:290
2． D'Souza RN, Bronckers ALJJ, Happonen RP, et al. Developmental expression of a.53kD dentin sialoprotein in rat tooth germs. J Histochem Cytochem, 1992, 40:359
3． Bronckers ALJJ, D'Souza RN, Butler WT, et al. Dentin sialoprotein: biosynthesis and denvelopmental appearance in rat tooth germs in comparison with amelogenins, osteocalcin and collagen type-1 . Cell Tissue Res, 1993, 272:237
4． Be gue-Kirn C, Krebshach PH, Bartlett JD, et al. Dentin sialoprotein, dentin phosphoprotein, enamelysin and ameloblastin: tooth-specific molecules that are distinctively expressed during murine dental differentiation. Eur J Oral Sci, 1998, 106: 963
5． BEgue-kirnC, Ruch JV, Ridall AL, et al. Comparative analysis of mouse DSP and DPP expression in odontoblasts, preameloblasts, and experimentally induced odontoblast-like
cells. Eur J Oral Sci, 1998, 106:254
6． Butler WT, Bhown M, Brunn.JC, et al. Isolation, characterization and immunolocalization of a 53kDa dentin sialoprotein (DSP). Matrix, 1992, 12:343
7． Ritchie HH, Berry JE, Somerman MJ, et al. Dentin sialoprotein (DSP) transcripts: developmentally sustained expression in odontoblasts and transient expression in ameloblasts. Eur J Oral Sci, 1997, 105:405
8． Ritchie HH, Hou H, Veis A, et al. Cloning and sequence determination of rat dentin sialoprotein, a noval dentin protein. J Biol Chem, 1994, 269:3 698
9． William T Butler. Dentin matrix proteins and dentinogenesis. Connect Tissue Res, 1995, 33:59-65[381-387]
10． Boskey AL. Osteopontin and related phosphorylated sialoproteins effects on mineralization. Ann NY Acad Sci, 1995, 760:249
11． Ritchie HH, Li X. A noval rat dentin mRNA coding only for dentin sialoprotein. Eur J Oral Sci, 2001, 109:342
12． Veis A, Perry A. The phosphoprotein of the dentin matrix. Biochemistry, 1967, 6(8) :2 409
13． Dickson IR, Dimuzio MT, Volpin D, et al. The extraction of phosphoproteins from bovine dentin. Calcif Tissue Res, 1975, 19(1) :51
14． Jonsson M, Fredriksson S. Isoelectric focusing of the phosphoprotein of rat-incisor dentin in ampholine and acid pH gradients. Evidence for carrier ampholyte-protein complexes. J Chromatogr, 1978, 157:234
15． MacDougall M, Zeichner-David M, Slavkin HC. Production and characterization of antibodies against murine dentine phosphoprotein. Biochem J, 1985, 232(2) :493
16． Stetler-Stevenson WG, Veis A. Bovine dentin phosphophoryn: composition and molecular weight. Biochemistry, 1983, 22(18) :4 326
17． Butler WT, Bhown M, DiMuzio MT, et al. Multiple forms of rat dentin phosphoproteins. Arch Biochem Biophys, 1983, 225(1) :178
18． Jontell M, Pertoft H, Linde A. Disagreement in molecular weight determinations of dentin phosphoprotein. Biochim Biophys Acta, 1982, 705(3) :315
19． Chang SR, Chiego D Jr, Clarkson BH. Characterization and identification of a human dentin phosphophoryn. Calcif Tissue Int, 1996, 59(3) : 149
20． DiMuzio MT, Bhown M, Butler WT. The biosynthesis of dentin phosphophoryns by rat incisor odontoblasts in organ culture. Calcif Tissue Int, 1985, 37(3) :242
21． Dimuzio MT, Veis A. The biosynthesis of phosphophoryns and dentin collagen in the continuously erupting rat incisor. J Biol Chem, 1978, 253(19) :6 845
22． Vries IGD, Quatier E, Steireghem V, et al. Characterization and immunocytochemical localization of dentin phosphoprotein in rat and bovine teeth. Archs Oral Biol, 1986, 31:74
23． Jontell M, Linde A. Non-collagenous proteins of predentine from dentinogenically active bovine teeth. Biochem J, 1983, 214(3) :769
24． Tagaki Y, Veis A. Isolation of phosphophoryn from human dentin organic matrix. Calcif Tissue Int, 1984,36:259
25． Gorter de Vries I, Quartier E, Van Steirteghem A, et al. Characterization and immunocytochemical localization of dentine phosphoprotein in rat and bovine teeth. Arch Oral Biol, 1986, 31(1) :57
26． Rahima M, Tsay TG, Andujar M, et al. Localization of phosphophoryn in rat incisor dentin using immunocytochemical techniques. J Histochem Cytochem, 1988, 36(2) : 153
27． Marsh ME. Self-association of calcium and magnesium complexes of dentin phosphophoryn. Biochemistry, 1989, 28(1) :339
28． Zanetti M, de Bernard B, Jontell M, et al. Ca2+-binding studies of the phosphoprotein from rat-incisor dentine. Eur J Biochem, 1981, 113(3) :541
29． Kuboki Y, Fujisawa R, Tsuzaki M, et al. Presence of lysinoalanine and histidinoalanine in bovine dentin phosphoprotein. Calcif Tissue Int, 1984, 36(1) : 126
30． Stetler-Stevenson WG, Veis A. Type I collagen shows a specific binding affinity for bovine dentin phosphophoryn. Calcif Tissue Int, 1986,38(3) : 135
31． Fujisawa R, Kuboki Y. Affinity of bone sialoprotein and several other bone and dentin acidic proteins to collagen fibrils. Calcif Tissue Int, 1992, 51(6) :438
32． Traub W, Arad T, Weiner S. Origin of mineral crystal growth in collagen fibrils. Matrix,
1992, 12(4) :251
33． Lussi A, Crenshaw MA, Linde A. Induction and inhibition of hydroxyapatite formation by ral dentine phosphoprotein in vitro. Arch Oral Biol, 1988, 33(9) :685
34． Linde A, Lussi A, Crenshaw MA. Mineral induction by immobilized polyanionic proteins. Calcif Tissue Int, 1989, 44(4) : 286
35． Boskey AL, Maresca M, Doty S, et al. Concentration-dependent effects of dentin phosphophoryn in the regulation of in vitro hydroxyapatite formation and growth. Bone Miner, 1990, 11(1) :55
36． Traub W, Jodalkin A, Arad T, et al. Dentin phosphophoryn binding to collagen fibrils. Matrix, 1992, 12:197
37． Ritchie HH, Wang LH.Sequence determination of an extremely acidic rat dentin phosphoprotein. J Biol Chem, 1996,271:21 695
38． Linde A, Bhown M, Butler WT. Related Articles Noncollagenous proteins of dentin. A re-examination of proteins from rat incisor dentin utilizing techniques to avoid artifacts. J Biol Chem, 1980, 255(12) :5 931
39． Veis A, Wei K, Sfeir C, et al. Properties of the (DSS)n triplet repeat domain of rat dentin phosphophoryn. Eur J Oral Sci, 1998, 106 (Suppl1) :234
40． Gu K, Chang SR, Slaven MS, et al. Human dentin phosphophoryn nucleotide and amino acid sequence. Eur J Oral Sci, 1998, 106(6) : 1 043．
41． Ritchie HH, Wang LH. A mammalian bicistronic transcript encoding two dentin-specific proteins. Biochem Biophy Res Commun, 1997, 231:425
42． MacDougall Mary, Simmons Darrin, Luan Xianghong, et al. Dentin phosphoprotein and dentin sailoprotein are cleavage products expressed from a single transcript coded by a gene on human chromosome 4． J Bio Chem, 1997, 272: 835
43． Gu K, Chang S, Ritchie HH, et al. Molecular cloning of a human dentin sialophophoprotein gene. Eur J Oral Sci, 2000,108:35
44． Linde A, Goldberg M. Dentinogenesis. Crit Rev Oral Biol Med, 1993, 4: 679
45． Hohling HJ, Arnold S, Barckhaus RH, et al. Structural relationship between the primary
crystal formation and the matrix macromolecules in different hard tissues. Discussion of a general principle. Connect Tissue Res, 1995, 33:171
46． Butler WT and Ritchie HH. The nature and functional significance of dentin exteacellular matrix protein. Int J Dev Bio, 1995, 39:169
47． Weinstock M, Leblond CP. Radlioautographic visualization of the deposition of a phosphoprotein at the mineralization front in the dentin of the rat incisor. J Cell Biol, 1973, 56(3) :838
48． Curley-Joseph J, Veis A. The nature of covalent complexes of phosphoproteins with collagen in the bovine dentin matrix. J Dent Res, 1979, 58(6) : 1 625
49． Suzuki Y, Yamaguchi A, Ikeda T, et al. In situ phosphorylation of bone and dentin proteins by the casein kinase II-like enzyme. J Dent Res, 1998,77(10) :1799
50． Veis A. Mineral-matrix interactions in bone and dentin. J Bone Miner Res, 1993, 8 (Suppl 2) : S493．
51． Satoyoshi M, Koizumi T, Teranaka T, et al. Extracellular processing of dentin matrix protein in the mineralizing odontoblast culture. Calcif Tissue Int, 1995, 57(3) :237
52． Mikuni-Takagaki Y, Kakai Y, Satoyoshi M, et al. Matrix mineralization and the differentiation of osteocyte-like cells in culture. J Bone Miner Res, 1995, 10(2) :231
53． Mikuni-Takagaki Y, Glimcher MJ. Post-translational processing of chicken bone phosphoproteins. Identification of the bone phosphoproteins of embryonic tibia. Biochem J, 1990, 268(3) :585
54． Wu CB, Pelech SL, Veis A. The in vitro phosphorylation of the native rat incisor dentin phosphophoryns. J Biol Chem, 1992,267(23) : 16588
55． Ackerman P, Glover CV, Osheroff N. Stimulation of casein kinase II by epidermal growth factor: relationship between the physiological activity of the kinase and the phosphorylation state of its beta subunit. Proc Natl Acad Sci USA, 1990, 87(2) : 821
56． Boskey AL. The role of extracellular matrix components in dentin mineralization. Crit Rev Oral Biol Med, 1991, 2(3) :369
57． Mann S. Molecular tectonics in biomineralization and biomimetic materials chemistry.
Nature, 1993, 365:499
58． Plate U, Hohling HJ, Reimer L, et al. Analysis of the calcium distribution in predentine by EELS and of the early crystal formation in dentine by ESI and ESD. J Microsc, 1992, 166:329
59． Wiesmann HP, Plate U, Hohling HJ, et al. Analysis of early hard tissue formation in dentine by energy dispersive X-ray microanalysis and energy-filtering transmission electron microscopy. Scanning Microsc, 1993, 7(2) :711．
60． Nelson DG, Barry JC. High resolution electron microscopy of nonstoichiometric apatite crystals. Anat Rec, 1989, 224(2) :265
61． Cuisinier F, Steuer P, Senger B, et al. Human amelogenesis: High resolution electron microscopy of nanometer-sized particles. Cell Tissue Res, 1993, 273:175
62． Plate U, Arnold S, Reimer L, Hohling HJ, et al. Investigation of the early mineralization on collagen in dentin by quantitative electron spectrocopic diffraction (ESD). Cell Tissue Res, 1994, 278:543
63． Cuisinier F, Steuer P, Brisson A, et al. High resolusion electron microscopy study of crystal growth mechanisms in chicken bone composites. J Cryst Growth, 1995, 156:443
64． Diekwisch G, Berman J, Gentner S, et al. Initial enamel crystlals are not associated with mineralized dentin. Cell Tissue Res, 1995, 279:149
65． Houlle E, Voegel JC, Schultz P, et al. High resolution electron microscopy: structure and growth mechanisms ofhuman dentin crystals. J Dent Res, 1997,76(3) :895
66． Bres EF, Steuer P, Voegel JC et al. Observation of the loss of the hydroxyapatite sixford symmetry in a human fetal tooth enamel crystal. J Microsc, 1993, 170:147
67． van den Bos T, Steinfort J, Beertsen W. Effect of bound phosphoproteins and other organic phosphates on alkaline phosphatase-induced mineralization of collagenous matrices in vitro. Bone Miner, 1993, 23(2) :81
68． Witkop CJ. Hereditary defects in enamel and dentin. Acta Genet, 1957, 7:236
69． Conneally PM, Bixler D, Horton-Kelly, et al. Confirmation of linkage between dentinogenesis imperfecta and Gc. Cytogenet Cell Genet, 1987, 42:438
70． Crosby AH, Scherpbier-Heddema T, Wijmenga C, et al. Genetic mapping of the dentinogenesis imperfecta type II locus. Am J Hum Genet, 1995, 57(4) :832
71． MacDougall M, DuPont BR, Simmons D, et al. Assignment of DMP1 to human chromosome 4 band 4q21 by in situ hybridization. Cytogenet Cell Genet, 1996, 74:189
72． Aplin HM, Hirst KL, Crosby AH, et al. Mapping of the human dentin matrix acidic phosphoprotein gene (DMP1) to the dentinogenesis imperfecta type II critical region at chromosome 4q21． Genomics, 1995, 30:347
73． MacDougall M, Simmons D, Luan X, et al. Assignment of dentin sialophosphoprotein to human chromosome 4 band q21． 3 by in situ hybridization. Cytogenet Cell Genet, 1997, 79(1-2) : 121
74． MacDougall M. Refined mapping of the human dentin sialophosphoprotein (DSPP) gene within the critical dentinogenesis imperfecta type II and dentin dysplasia type II loci. Eur J Oral Sci, 1998, 106(suppl 1) :227
75． 王英.遗传性牙本质发育不全II型致病基因的定位与克隆，第四军医大学博士学位论文, 2001
76． Zhang X, Zhao J, Li C, et al. DSPP mutation in dentinogenesis imperfecta shields type II. Nat Genet, 2001,27:151
77． Takagi Y, Veis A. Matrix protein differences between human normal and dentinogenesis imperfecta dentin. In: Veis A (ed). The Chemistry and biology of mineralized connective tissues. Elsever/North-Holland, New York, 1981, 233
78． Takagi Y, Veis A, Sauk JJ. Relation of minerlization defects in collagen matrices to non-collagen component: identification of a molecular defect in dentinogenesis imperfecta. Clin Orthop Related Res, 1983, 176:282
79． Takagi Y, Sasaki. Histological distribution of phosphophoryn in normal and pathological human dentins. J Oral Pathol, 1986, 15:463
80． Ritchie HH, Ritchie DG, Wang LH. Six decades of dentinogenesis research: Historical and prospective views on phosphophoryn and dentin sialoprotein. Eur J Oral Sci, 1998, 106(suppl1) :211
81．张莹，张郁．牙髓损伤修复的机制．国外医学口腔医学分册，1997，26(4)：241
82. Paterson RC. Pulp response in sound and carious teeth: a pilot study. Oral Surg Oral Med Oral Pathol, 1981, 51:209
83. Cassidy N, Fahey M, Prime SS, et al. Comparative analysis of transforming growth factor-beta isoforms 1-3 in human and rabbit dentin matrices. Arch Oral Biol, 1997,42(3):219
84. Smith AJ,Tobias RS, Cassidy N, et al. Odontoblast stimulation in ferrets by dentin matrix components. Arch Oral Biol, 1994, 39:13
85. Nakashima M. An ultrastructure study of the differentiationts of mesenchymal cells in implants of allogeneic dentine matrix on the amputated dental pulp of the dog. Arch Oral Biol, 1990, 35(4):277
86. Tziafas D, Alvanou A, Panagiotakopoulos N, et al. Induction of odontoblast-like cell differentiation in dog dental pulps after in vivo implantation of dentin matrix components. Arch Oral Biol, 1995, 40(10):883
87. Nakashima M. Dentin induction by implants of autolyzed antigen-extracted allogeneic dentin on amputated pulps of dogs. Endod Dent Traumatol, 1989, 5:279
88. Smith A J, Cassidy N, Perry H, et al. Reactionary dentinogenesis. Int J Dev Biol, 1995, 39:273
89. Lussi A and Linde A. Mineral induction in vivo by dentin proteins. Caries Res, 1993, 27:241
90. van den Bos T, Beertsen W. Bound phosphoproteins enhance mineralization of alkaline phosphatase-collagen complexes in vivo. J Bone Miner Res, 1994, 9(8): 1 205
91．欧阳勇，李玉晶，宿颖．人牙本质磷蛋白的提取与特性分析．中华口腔医学杂志，1999，34(2)：112
92．欧阳勇，李玉晶，刘晓勇等．人牙本质磷蛋白对小型猪修复性牙本质形成作用的实验研究．中华口腔医学杂志，1999，34(5)：295
93．张蓉．牙本质涎磷蛋白在牙齿发育、矿化及牙髓损伤修复中作用的研究．第四军医大学博士学位论文，2001
2． D'Souza RN, Bronckers ALJJ, Happonen RP, et al. Developmental expression of a.53kD dentin sialoprotein in rat tooth germs. J Histochem Cytochem, 1992, 40:359
3． Bronckers ALJJ, D'Souza RN, Butler WT, et al. Dentin sialoprotein: biosynthesis and denvelopmental appearance in rat tooth germs in comparison with amelogenins, osteocalcin and collagen type-1 . Cell Tissue Res, 1993, 272:237
4． Be gue-Kirn C, Krebshach PH, Bartlett JD, et al. Dentin sialoprotein, dentin phosphoprotein, enamelysin and ameloblastin: tooth-specific molecules that are distinctively expressed during murine dental differentiation. Eur J Oral Sci, 1998, 106: 963
5． BEgue-kirnC, Ruch JV, Ridall AL, et al. Comparative analysis of mouse DSP and DPP expression in odontoblasts, preameloblasts, and experimentally induced odontoblast-like
cells. Eur J Oral Sci, 1998, 106:254
6． Butler WT, Bhown M, Brunn.JC, et al. Isolation, characterization and immunolocalization of a 53kDa dentin sialoprotein (DSP). Matrix, 1992, 12:343
7． Ritchie HH, Berry JE, Somerman MJ, et al. Dentin sialoprotein (DSP) transcripts: developmentally sustained expression in odontoblasts and transient expression in ameloblasts. Eur J Oral Sci, 1997, 105:405
8． Ritchie HH, Hou H, Veis A, et al. Cloning and sequence determination of rat dentin sialoprotein, a noval dentin protein. J Biol Chem, 1994, 269:3 698
9． William T Butler. Dentin matrix proteins and dentinogenesis. Connect Tissue Res, 1995, 33:59-65[381-387]
10． Boskey AL. Osteopontin and related phosphorylated sialoproteins effects on mineralization. Ann NY Acad Sci, 1995, 760:249
11． Ritchie HH, Li X. A noval rat dentin mRNA coding only for dentin sialoprotein. Eur J Oral Sci, 2001, 109:342
12． Veis A, Perry A. The phosphoprotein of the dentin matrix. Biochemistry, 1967, 6(8) :2 409
13． Dickson IR, Dimuzio MT, Volpin D, et al. The extraction of phosphoproteins from bovine dentin. Calcif Tissue Res, 1975, 19(1) :51
14． Jonsson M, Fredriksson S. Isoelectric focusing of the phosphoprotein of rat-incisor dentin in ampholine and acid pH gradients. Evidence for carrier ampholyte-protein complexes. J Chromatogr, 1978, 157:234
15． MacDougall M, Zeichner-David M, Slavkin HC. Production and characterization of antibodies against murine dentine phosphoprotein. Biochem J, 1985, 232(2) :493
16． Stetler-Stevenson WG, Veis A. Bovine dentin phosphophoryn: composition and molecular weight. Biochemistry, 1983, 22(18) :4 326
17． Butler WT, Bhown M, DiMuzio MT, et al. Multiple forms of rat dentin phosphoproteins. Arch Biochem Biophys, 1983, 225(1) :178
18． Jontell M, Pertoft H, Linde A. Disagreement in molecular weight determinations of dentin phosphoprotein. Biochim Biophys Acta, 1982, 705(3) :315
19． Chang SR, Chiego D Jr, Clarkson BH. Characterization and identification of a human dentin phosphophoryn. Calcif Tissue Int, 1996, 59(3) : 149
20． DiMuzio MT, Bhown M, Butler WT. The biosynthesis of dentin phosphophoryns by rat incisor odontoblasts in organ culture. Calcif Tissue Int, 1985, 37(3) :242
21． Dimuzio MT, Veis A. The biosynthesis of phosphophoryns and dentin collagen in the continuously erupting rat incisor. J Biol Chem, 1978, 253(19) :6 845
22． Vries IGD, Quatier E, Steireghem V, et al. Characterization and immunocytochemical localization of dentin phosphoprotein in rat and bovine teeth. Archs Oral Biol, 1986, 31:74
23． Jontell M, Linde A. Non-collagenous proteins of predentine from dentinogenically active bovine teeth. Biochem J, 1983, 214(3) :769
24． Tagaki Y, Veis A. Isolation of phosphophoryn from human dentin organic matrix. Calcif Tissue Int, 1984,36:259
25． Gorter de Vries I, Quartier E, Van Steirteghem A, et al. Characterization and immunocytochemical localization of dentine phosphoprotein in rat and bovine teeth. Arch Oral Biol, 1986, 31(1) :57
26． Rahima M, Tsay TG, Andujar M, et al. Localization of phosphophoryn in rat incisor dentin using immunocytochemical techniques. J Histochem Cytochem, 1988, 36(2) : 153
27． Marsh ME. Self-association of calcium and magnesium complexes of dentin phosphophoryn. Biochemistry, 1989, 28(1) :339
28． Zanetti M, de Bernard B, Jontell M, et al. Ca2+-binding studies of the phosphoprotein from rat-incisor dentine. Eur J Biochem, 1981, 113(3) :541
29． Kuboki Y, Fujisawa R, Tsuzaki M, et al. Presence of lysinoalanine and histidinoalanine in bovine dentin phosphoprotein. Calcif Tissue Int, 1984, 36(1) : 126
30． Stetler-Stevenson WG, Veis A. Type I collagen shows a specific binding affinity for bovine dentin phosphophoryn. Calcif Tissue Int, 1986,38(3) : 135
31． Fujisawa R, Kuboki Y. Affinity of bone sialoprotein and several other bone and dentin acidic proteins to collagen fibrils. Calcif Tissue Int, 1992, 51(6) :438
32． Traub W, Arad T, Weiner S. Origin of mineral crystal growth in collagen fibrils. Matrix,
1992, 12(4) :251
33． Lussi A, Crenshaw MA, Linde A. Induction and inhibition of hydroxyapatite formation by ral dentine phosphoprotein in vitro. Arch Oral Biol, 1988, 33(9) :685
34． Linde A, Lussi A, Crenshaw MA. Mineral induction by immobilized polyanionic proteins. Calcif Tissue Int, 1989, 44(4) : 286
35． Boskey AL, Maresca M, Doty S, et al. Concentration-dependent effects of dentin phosphophoryn in the regulation of in vitro hydroxyapatite formation and growth. Bone Miner, 1990, 11(1) :55
36． Traub W, Jodalkin A, Arad T, et al. Dentin phosphophoryn binding to collagen fibrils. Matrix, 1992, 12:197
37． Ritchie HH, Wang LH.Sequence determination of an extremely acidic rat dentin phosphoprotein. J Biol Chem, 1996,271:21 695
38． Linde A, Bhown M, Butler WT. Related Articles Noncollagenous proteins of dentin. A re-examination of proteins from rat incisor dentin utilizing techniques to avoid artifacts. J Biol Chem, 1980, 255(12) :5 931
39． Veis A, Wei K, Sfeir C, et al. Properties of the (DSS)n triplet repeat domain of rat dentin phosphophoryn. Eur J Oral Sci, 1998, 106 (Suppl1) :234
40． Gu K, Chang SR, Slaven MS, et al. Human dentin phosphophoryn nucleotide and amino acid sequence. Eur J Oral Sci, 1998, 106(6) : 1 043．
41． Ritchie HH, Wang LH. A mammalian bicistronic transcript encoding two dentin-specific proteins. Biochem Biophy Res Commun, 1997, 231:425
42． MacDougall Mary, Simmons Darrin, Luan Xianghong, et al. Dentin phosphoprotein and dentin sailoprotein are cleavage products expressed from a single transcript coded by a gene on human chromosome 4． J Bio Chem, 1997, 272: 835
43． Gu K, Chang S, Ritchie HH, et al. Molecular cloning of a human dentin sialophophoprotein gene. Eur J Oral Sci, 2000,108:35
44． Linde A, Goldberg M. Dentinogenesis. Crit Rev Oral Biol Med, 1993, 4: 679
45． Hohling HJ, Arnold S, Barckhaus RH, et al. Structural relationship between the primary
crystal formation and the matrix macromolecules in different hard tissues. Discussion of a general principle. Connect Tissue Res, 1995, 33:171
46． Butler WT and Ritchie HH. The nature and functional significance of dentin exteacellular matrix protein. Int J Dev Bio, 1995, 39:169
47． Weinstock M, Leblond CP. Radlioautographic visualization of the deposition of a phosphoprotein at the mineralization front in the dentin of the rat incisor. J Cell Biol, 1973, 56(3) :838
48． Curley-Joseph J, Veis A. The nature of covalent complexes of phosphoproteins with collagen in the bovine dentin matrix. J Dent Res, 1979, 58(6) : 1 625
49． Suzuki Y, Yamaguchi A, Ikeda T, et al. In situ phosphorylation of bone and dentin proteins by the casein kinase II-like enzyme. J Dent Res, 1998,77(10) :1799
50． Veis A. Mineral-matrix interactions in bone and dentin. J Bone Miner Res, 1993, 8 (Suppl 2) : S493．
51． Satoyoshi M, Koizumi T, Teranaka T, et al. Extracellular processing of dentin matrix protein in the mineralizing odontoblast culture. Calcif Tissue Int, 1995, 57(3) :237
52． Mikuni-Takagaki Y, Kakai Y, Satoyoshi M, et al. Matrix mineralization and the differentiation of osteocyte-like cells in culture. J Bone Miner Res, 1995, 10(2) :231
53． Mikuni-Takagaki Y, Glimcher MJ. Post-translational processing of chicken bone phosphoproteins. Identification of the bone phosphoproteins of embryonic tibia. Biochem J, 1990, 268(3) :585
54． Wu CB, Pelech SL, Veis A. The in vitro phosphorylation of the native rat incisor dentin phosphophoryns. J Biol Chem, 1992,267(23) : 16588
55． Ackerman P, Glover CV, Osheroff N. Stimulation of casein kinase II by epidermal growth factor: relationship between the physiological activity of the kinase and the phosphorylation state of its beta subunit. Proc Natl Acad Sci USA, 1990, 87(2) : 821
56． Boskey AL. The role of extracellular matrix components in dentin mineralization. Crit Rev Oral Biol Med, 1991, 2(3) :369
57． Mann S. Molecular tectonics in biomineralization and biomimetic materials chemistry.
Nature, 1993, 365:499
58． Plate U, Hohling HJ, Reimer L, et al. Analysis of the calcium distribution in predentine by EELS and of the early crystal formation in dentine by ESI and ESD. J Microsc, 1992, 166:329
59． Wiesmann HP, Plate U, Hohling HJ, et al. Analysis of early hard tissue formation in dentine by energy dispersive X-ray microanalysis and energy-filtering transmission electron microscopy. Scanning Microsc, 1993, 7(2) :711．
60． Nelson DG, Barry JC. High resolution electron microscopy of nonstoichiometric apatite crystals. Anat Rec, 1989, 224(2) :265
61． Cuisinier F, Steuer P, Senger B, et al. Human amelogenesis: High resolution electron microscopy of nanometer-sized particles. Cell Tissue Res, 1993, 273:175
62． Plate U, Arnold S, Reimer L, Hohling HJ, et al. Investigation of the early mineralization on collagen in dentin by quantitative electron spectrocopic diffraction (ESD). Cell Tissue Res, 1994, 278:543
63． Cuisinier F, Steuer P, Brisson A, et al. High resolusion electron microscopy study of crystal growth mechanisms in chicken bone composites. J Cryst Growth, 1995, 156:443
64． Diekwisch G, Berman J, Gentner S, et al. Initial enamel crystlals are not associated with mineralized dentin. Cell Tissue Res, 1995, 279:149
65． Houlle E, Voegel JC, Schultz P, et al. High resolution electron microscopy: structure and growth mechanisms ofhuman dentin crystals. J Dent Res, 1997,76(3) :895
66． Bres EF, Steuer P, Voegel JC et al. Observation of the loss of the hydroxyapatite sixford symmetry in a human fetal tooth enamel crystal. J Microsc, 1993, 170:147
67． van den Bos T, Steinfort J, Beertsen W. Effect of bound phosphoproteins and other organic phosphates on alkaline phosphatase-induced mineralization of collagenous matrices in vitro. Bone Miner, 1993, 23(2) :81
68． Witkop CJ. Hereditary defects in enamel and dentin. Acta Genet, 1957, 7:236
69． Conneally PM, Bixler D, Horton-Kelly, et al. Confirmation of linkage between dentinogenesis imperfecta and Gc. Cytogenet Cell Genet, 1987, 42:438
70． Crosby AH, Scherpbier-Heddema T, Wijmenga C, et al. Genetic mapping of the dentinogenesis imperfecta type II locus. Am J Hum Genet, 1995, 57(4) :832
71． MacDougall M, DuPont BR, Simmons D, et al. Assignment of DMP1 to human chromosome 4 band 4q21 by in situ hybridization. Cytogenet Cell Genet, 1996, 74:189
72． Aplin HM, Hirst KL, Crosby AH, et al. Mapping of the human dentin matrix acidic phosphoprotein gene (DMP1) to the dentinogenesis imperfecta type II critical region at chromosome 4q21． Genomics, 1995, 30:347
73． MacDougall M, Simmons D, Luan X, et al. Assignment of dentin sialophosphoprotein to human chromosome 4 band q21． 3 by in situ hybridization. Cytogenet Cell Genet, 1997, 79(1-2) : 121
74． MacDougall M. Refined mapping of the human dentin sialophosphoprotein (DSPP) gene within the critical dentinogenesis imperfecta type II and dentin dysplasia type II loci. Eur J Oral Sci, 1998, 106(suppl 1) :227
75． 王英.遗传性牙本质发育不全II型致病基因的定位与克隆，第四军医大学博士学位论文, 2001
76． Zhang X, Zhao J, Li C, et al. DSPP mutation in dentinogenesis imperfecta shields type II. Nat Genet, 2001,27:151
77． Takagi Y, Veis A. Matrix protein differences between human normal and dentinogenesis imperfecta dentin. In: Veis A (ed). The Chemistry and biology of mineralized connective tissues. Elsever/North-Holland, New York, 1981, 233
78． Takagi Y, Veis A, Sauk JJ. Relation of minerlization defects in collagen matrices to non-collagen component: identification of a molecular defect in dentinogenesis imperfecta. Clin Orthop Related Res, 1983, 176:282
79． Takagi Y, Sasaki. Histological distribution of phosphophoryn in normal and pathological human dentins. J Oral Pathol, 1986, 15:463
80． Ritchie HH, Ritchie DG, Wang LH. Six decades of dentinogenesis research: Historical and prospective views on phosphophoryn and dentin sialoprotein. Eur J Oral Sci, 1998, 106(suppl1) :211
81．张莹，张郁．牙髓损伤修复的机制．国外医学口腔医学分册，1997，26(4)：241
82. Paterson RC. Pulp response in sound and carious teeth: a pilot study. Oral Surg Oral Med Oral Pathol, 1981, 51:209
83. Cassidy N, Fahey M, Prime SS, et al. Comparative analysis of transforming growth factor-beta isoforms 1-3 in human and rabbit dentin matrices. Arch Oral Biol, 1997,42(3):219
84. Smith AJ,Tobias RS, Cassidy N, et al. Odontoblast stimulation in ferrets by dentin matrix components. Arch Oral Biol, 1994, 39:13
85. Nakashima M. An ultrastructure study of the differentiationts of mesenchymal cells in implants of allogeneic dentine matrix on the amputated dental pulp of the dog. Arch Oral Biol, 1990, 35(4):277
86. Tziafas D, Alvanou A, Panagiotakopoulos N, et al. Induction of odontoblast-like cell differentiation in dog dental pulps after in vivo implantation of dentin matrix components. Arch Oral Biol, 1995, 40(10):883
87. Nakashima M. Dentin induction by implants of autolyzed antigen-extracted allogeneic dentin on amputated pulps of dogs. Endod Dent Traumatol, 1989, 5:279
88. Smith A J, Cassidy N, Perry H, et al. Reactionary dentinogenesis. Int J Dev Biol, 1995, 39:273
89. Lussi A and Linde A. Mineral induction in vivo by dentin proteins. Caries Res, 1993, 27:241
90. van den Bos T, Beertsen W. Bound phosphoproteins enhance mineralization of alkaline phosphatase-collagen complexes in vivo. J Bone Miner Res, 1994, 9(8): 1 205
91．欧阳勇，李玉晶，宿颖．人牙本质磷蛋白的提取与特性分析．中华口腔医学杂志，1999，34(2)：112
92．欧阳勇，李玉晶，刘晓勇等．人牙本质磷蛋白对小型猪修复性牙本质形成作用的实验研究．中华口腔医学杂志，1999，34(5)：295
93．张蓉．牙本质涎磷蛋白在牙齿发育、矿化及牙髓损伤修复中作用的研究．第四军医大学博士学位论文，2001