【作者】 张莹；

【导师】 史俊南；

【作者基本信息】 第四军医大学 ， 口腔内科学， 2002， 博士

【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更多还原

【Abstract】 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 hDSPIn the present study, total RNA was extracted from the tooth germs of alegally 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 hDSPForeign 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 forfurther functional study.3. Functional study of h DSPThe 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更多还原