| 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...
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