【作者】 高杰；

【导师】 吴补领；

【作者基本信息】 第四军医大学 ， 口腔临床医学， 2002， 硕士

【摘要】 牙本质非胶原蛋白(NCPs)在牙齿矿化过程中的作用一直是牙髓生物学的研究热点。应用组织学、生物化学和分子生物学等技术，从细胞水平、蛋白水平以至分子水平，已对其进行了大量的研究，使我们对牙本质矿化的过程和机理有了深入的理解。但目前的研究多集中于牙本质特异性蛋白，而对矿化组织特异性蛋白的研究相对较少。至于矿化组织特异性蛋白的确切生物学功能；它们之间以及它们与其他非胶原蛋白之间的相互作用；其在牙齿发育和骨组织发育中的作用：它们如何调节矿化过程，与牙本质发育不全症的关系等难题，仍有待我们作进一步深入研究。 牙本质基质蛋白1(dentin matrix protein 1 DMP1)是矿化组织特异性蛋白的重要成员之一，它富含天门冬氨酸、丝氨酸和谷氨酸，是一种分泌性磷酸化蛋白，与BAG-75有高度同源性，与骨桥素有部分同源性，它的组成介于骨酸性糖蛋白和牙齿磷酸化蛋白之间。研究表明：过表达DMP1基因的未分化间充质细胞可向成牙本质细胞样细胞分化，并可以使其在分化的早期阶段表达ALP、Cbfa1、BMP4，在分化的末期阶段表达BSP、OCN、OPN。而且随着DMP1表达量的增加，DMP2和DSP的表达量也增加。而用酪蛋白激酶Ⅱ抑制剂阻断DMP1磷酸化，DMP2、DSP、Cbfa1、OCN的表达未见明显变化。同时，过表达DMP1 京回军巨大攀双士学位论文 基因的成牙本质样细胞可以促进细胞内矿化小结的形成和增大。由此可推测， 体内 DMP在未分化间充质细胞向成牙本质细胞分化以及骨和牙齿硬组织形成 过程中起着非常重要的作用。 目前关于 DMP的研究集中于分子生物学结构和功能。由于缺乏良好的工 召 具，其在蛋白水平的表达和功能研究较少。为获得良好的研究工具，以揭示 DMPI蛋白在组织矿化过程中所起的作用，本实验组制备了抗大鼠DMPI的单克 隆抗体，并初步观察其生物学功能。 主要研究内容及结果如下： 一．大鼠牙本质基质蛋白1对体外培养的人牙髓细胞增殖和碱性磷酸酶活性 的影响 利用MTT法研究牙髓细胞的细胞增殖情况，并研究DMPI对牙髓细胞内碱性 磷酸酶活性的影响。DMPI的浓度为0．05ty／ml、0．fly／ml、0．sug／ml、互W／ml 和5％／ml。MTT实验刺激时间分别为id、3d、sd，结果显示：id组与对照组比 较无明显差异；3d和sd的sty砌组可促进人牙髓细胞增殖（P＜0．05），3d和 sd的其它各浓度组则与对照组无明显差异（P＞0．05）。ALP活性测定刺激时间 分别为3d、sd、7d，结果显示：细胞培养3d时，仅SPg砌1组可促进人牙髓细 胞从P活性（P（．05）；培养sd时fly／ml和sty／。l均可促进人牙髓细胞队P活 性（K0．05）；培养7d时促进作用进一步加强（k0．05）；sd组和7d组之间 无明显差异（P＞0．05）。结果提示：DMPI对牙髓细胞ALP活性的促进作用呈浓 度依赖性；高浓度DMP可以促进人牙髓细胞ALP活性，随着时间的延长，促进 ALP活性的作用也增强。由此表明DMPI在一定浓度下可促进牙髓细胞的增殖和 分化。 二．抗大鼠DMPI单克隆抗体的制备和鉴定 以大鼠重组DMP为抗原，免疫Balb／c ，J’鼠，通过B淋巴细胞杂交瘤技术， 即用B淋巴细胞与骨髓瘤细胞SP2／0进行细胞融合，利用间接ELISA法筛选 D ePeParo刃ent Of OPeOPerat。Dent。卿andblaoaontics 厂力山YU 3 章旧罩区大学呵士学位论文 阳性克隆，制备抗大鼠 DMP的单克隆抗体（MCAB），并对其性质进行鉴定。 结果：共获得2株单克隆抗体细胞株。dot hi。t结果显示2株抗体均与DMPI 和牙齿组织总蛋白能够反应，而与脑组织、肝脏、肾脏组织总蛋白无反应， 说明2株抗体均具有较好的特异性。从亲和常数可知ZH10的亲和力大于2C10 的亲和力。本实验首次获得了特异性较好、效价较高的大鼠 DMP单克隆抗体，4 为进一步研究DMPI的确切生理功能提供了良好的工具。 三．牙本质基质蛋白l在大鼠不同组织中的表达研究 选用处于不同发育时期SD大鼠牙胚和牙齿；不同发育时期成年鼠的股骨 以及成年大鼠肝脏、肾脏和脑组织。采用免疫组织化学的方法，对 DMP在大 鼠不同组织中的表达进行研究。结果显示：D惭 在出生后3d大鼠磨牙和切牙 （牙本质基质分泌期）的成牙本质细胞胞浆顶端阳性表达，表明 DMP可能以 分泌颗粒的形式集聚于成牙本质细胞胞浆顶端，最终沿成牙本质细胞突分泌更多还原

【Abstract】 Bone and dentin are mineralized tissues that resemble each other in the composition and mechanism of formation. In addition to a predominant collagenous matrix, bone and dentin contain non-collagenous proteins (NCPs). Since these NCPs are very acidic and are secreted into the extracellular matrix (ECM) during the formation and mineralization of these tissues, it is generally accepted that they play key biological roles in regulating the size and shape of the mineral crystal. Sialic acid-rich proteins, a category of mineralized tissue NCPs, include osteopontin(OPN), bone sialoprotein (BSP), bone acid glycoprotein-75 (BAG-75), dentin matrix protein 1(DMP1), and dentin sialoprotein (DSP). The precise functions of all these proteins are unknown.DMP1 is an acidic protein that was first cloned from the mineralized dentin matrix. A unique feature of DMP1 is that it is highly hydrophilic. DMP1 is rich with glutamic acid, aspartic acid, and serines. The serine residues are embedded in acidic sequences that make them good substrates for phosphorylation by casein kinase I and II. It could be postulated that DMP1, because of its high acidic nature, strong binding power to calcium, could initiate the nucleation process and turn on the entire cascade of regulated hydroxyapatite crystal growth. Although the precise function of DMP1 is yet not to be determined, in situ hybridization experiments have demonstrated that it is localized in both osteoblasts and odontoblasts. At the amino acid level, DMP1 has a single RGD domain, which was shown recently to be functional in cell attachment. A detailed understanding of the physiological function of DMP1 is still lacking, even though this protein has been well characterized. The main contents and results of the study are presented as follows: 1. The effects of DMP1 on the growth and alkaline phosphatase activityof cultured human dental pulp cells in vitroThe effects of DMP1 on the growth and alkaline phosphatase (ALP) activity of cultured human dental pulp cells were determined by MTT colorimetric assay and enzyme dynamical method. The concentrations of DMP1 added into culture medium were 0. 05M-g/ml, 0. iMg/ml, 0. SM-g/ml, lUg/ml and 5Mg/ml, which absence of DMP1 in culture medium was used as control. In MTT, Id, 3d and 5d were observed, the results shew that groups of Id there are no significant difference compared with control.5Mg/ral DMP1 in 3d and 5d stimulated the cell proliferation compared with control (p<0. 05), no diferrences were observed between 5Mg/ml DMP1 in groups of 3d and 5d. Other groups of DMP1 in 3d and 5d had no significant difference(p>0. 05)compared with control. Determining time of ALP activity were 3d, 5d and 7d. Results shew that among 3d groups, ALP activity was increased (p<0. 05) by DMP1 at 5Hg/ml compared with control. Among 5d groups, ALP activity was increased (p<0. 05) by DMP1 at iP-g/ml and 5^g/ml. Among 7d groups, the effect of DMP1 still increased. These results indicated that DMP1 could stimulate the growth of cultured dental pulp cell, and high concentrations of DMP1 could increase ALP activity of dental pulp cell. 2. Preparation and identification of monoclonal antibodies against ratrecombinant DMP1In order to prepare monoclonal antibodies (McABs) specifically against dentin matrix protein 1 (DMP1), we immunized Balb/c mice with rat recombined DMP1. Then McABs were produced by hybridomas derived from sp2/0 myeloma cells and spleen cells of immunized Balb/c mice. Results: Two hybridome cell lines, which stably secreted McABs against DMP1 were produced. Both McABs belonged to subclasses of IgGl. The results of dot hot shew that two McABs could specifically react with rat recombinant DMP1 and all dental proteins of rat, but could not react with any protein of rat brain, liver and kidney. The results indicated that two McABs obtained by hybridome technique had high affinity and were specific with DMP1. So far as we know, this was the first time about McABs of DMP1 reported. They could be of some help in the study更多还原