Molecular cloning of the factors that biologically accelerate reparative dentin formation

1.制备了携带β-半乳糖苷酶基因的重组腺病毒载体。用该载体培养小鼠成骨样细胞MC-3 T3-E1。结果表明，几乎所有的细胞都转染了β-半乳糖苷酶基因.已知因子（TGF-β 1和CTGF）在体内动物模型中的定位通过免疫组织化学染色在动物（wister大鼠）实验模型中证实了被认为参与修复性牙本质形成的TGF-β 1和CTGF的定位。结果，在离体牙2周内，观察到修复性牙本质样组织，并在这些组织周围观察到TGF-β 1和CTGF的强染色.促炎因子刺激体外培养的成牙本质样细胞中TGF-β 1和CTGF的基因表达本研究采用小鼠源性成牙本质样细胞MDPC-23。用IL-1 β和细菌LPS刺激细胞。RT-PCR检测CTGF和TGF-β 1基因表达的变化。结果显示，MDPC-23细胞呈CTGF和TGF-β 1基因的互补表达，并且在刺激后1天内，两种因子均使成牙本质细胞样细胞（MDPC-23）中CTGF基因的表达增加，而TGF-β 1基因的表达降低. TGF-β_1和CTGF对培养细胞的影响MTT法检测rCTGF和rTGF-β_1对细胞增殖的影响。rTGF-β 1呈剂量依赖性地抑制细胞增殖，而rCTGF的作用不明显。接下来，为了研究rCTGF对MDPC-23细胞钙化的影响，用含有rCTGF的培养基培养细胞。依次加入AA和b-GP可上调ALG活性，而rCTGF对ALG活性无明显影响。

1. Gene delivery to cultured cellsWe prepared recombinant adenovirus vector carrying beta-galactosidase gene. Mouse osteoblast-like cells, MC-3T3 -E1, were cultured with this vector. As a result, almost all cells were transfected with beta-galactosidase gene.2. Localization of known factors (TGF-beta 1 and CTGF) in vivo animal modelLocalization of TGF-beta 1, that is supposed to be involved in reparative dentinogenesis, and CTGF were confirmed with immunohistochemical staining in animal (wister rat) experimental model. As a result, in 2 weeks from tooth reduction reparative dentin-like tissues were observed, and strong staining of TGF-beta 1 and CTGF were observed around these tissues.3. Gene expression of TGF-beta 1 and CTGF in cultured cells stimulated with proinflammatory factorsMDPC-23, mouse-derived odontoblast-like cells were used in this study. The cells were stimulated with IL-1 beta and bacterial LPS. Changes in CTGF and TGF-b1 genes expression were examined by RT-PCR. As a result, MDPC-23 cells were expressing the CTGF and TGF-beta 1 genes coastitutively, and both factors increased CTGF gene expression and decreased TGF-b1 gene expression in the odontoblast-like cells (MDPC-23) within one-day period after stimulation.4. Effect of TGF-beta 1 and CTGF to cultured cellsThe effects of rCTGF and rTGF-beta 1 on cell proliferation were determined by the MTT assay. rTGF-beta 1 tended to decrease the proliferation dose-dependently, whereas effect of rCTGF was not evident. Next, to investigate the effects of rCTGF on calcification of MDPC-23 cells, cells were cultured with medium containing rCTGF. Sequential addition of AA and b-GP up-regulated the ALPase activity, while addition of rCTGF had no obvious effects.