Molecular cloning of the factors that biologically accelerate reparative dentin formation

1.基因导入培养细胞制备携带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-3T3-E1。结果，几乎所有的细胞都被导入了β-半乳糖苷酶基因2。已知因子(转化生长因子-β1和结缔组织生长因子)在动物模型中的定位在动物(Wister大鼠)实验模型中，免疫组织化学染色证实了与修复性牙本质形成有关的转化生长因子-β1和结缔组织生长因子的定位。结果显示，拔牙后2周可见修复性牙本质样组织，周围可见较强的转化生长因子-β1和结缔组织生长因子。促炎症因子MDPC-23、小鼠成牙本质细胞样细胞中转化生长因子-β1和结缔组织生长因子的基因表达用IL-1β和细菌内毒素刺激细胞。逆转录聚合酶链式反应检测CTGF和转化生长因子β1基因表达的变化。结果表明，MDPC-23细胞同时表达CTGF和TGF-β1基因，在刺激后1天内，这两种因子均可使成牙本质细胞样细胞(MDPC-23)CTGF基因表达增加，而TGF-β1基因表达降低。用四甲基偶氮唑盐比色法检测rCTGF和rTGF-β1对培养细胞增殖的影响。RTGF-β1呈剂量依赖性抑制细胞增殖，而rCTGF的作用不明显。为了研究rCTGF对MDPC-23细胞钙化的影响，用含有rCTGF的培养液培养MDPC-23细胞。顺次加入AA和b-gp可上调ALPase活性，而加入rCTGF则无明显影响。

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.