Role of Smad on Mineralization of Dental Pulp

我们研究了TNF-α对人牙髓（HDP）细胞矿化能力的影响。经TNF-α刺激的HDP细胞中BMP-2的RT-PCR产物的量增加。然而，在与TNF-α孵育的细胞上，ALP活性没有增加。而PDTC（NF-κB抑制剂）组和TNF-α与PDTC联合组ALP活性明显升高。此外，我们使用RT-PCR和western blot分析来检测TNF-α和PDTC对Smad 7表达的影响。Western blot结果显示，TNF-α可上调HDP细胞Smad 7蛋白表达，而PDTC和TNF-α联合作用后，HDP细胞Smad 7 mRNA和蛋白表达均较TNF-α明显降低。结果表明，NF-κB和Smad 7在TNF-α下调HDP细胞ALP活性中起重要作用。用1.0W的Ga-Al-As激光照射体外培养的HDP细胞500 s，茜素红S染色观察钙化结节的形成。激光照射组的激光照射量大于未照射的对照细胞。激光照射也增加了钙的产生。激光照射后ALP活性升高。Smad 1、Smad 7、BMP、ALP和骨钙素的mRNA表达在激光照射下更高。激光照射可抑制HDP细胞Smad 6 mRNA的表达。激光照射后，条件培养基中BMP-2和BMP-4的产量也较高。这些结果表明Smads和BMPs在激光照射HDP细胞后的ALP活性和钙化中起重要作用。

We examined effects of TNF-a on mineralization ability of human dental pulp (HDP) cells. The quantity of RT-PCR product for BMP-2 from the HDP cells stimulated by TNF-a is increased. However, ALP activity was not increased on the cells incubated with TNF-a. On the other hand, ALP activity was significantly increased on HDP cells treated with Ammonium Pyrrolidinedithiocarbamate (PDTC, NF-κB inhibitor) groups and combined supplementation of TNF-a and PDTC groups. Furthermore, we examined the effect of TNF-a and PDTC on Smad7 expression using RT-PCR and western blot analysis. Western blot analysis revealed that Smad7 protein in HDP cells were increased by TNF-a, but Smad7 mRNA and protein in HDP cells treated with supplementation of TNF-a and PDTC were decreased compared with TNF-a. These results suggest that NF-κB and Smad7 play an important role in the down regulation of ALP activity by TNF-a on HDP cells.We investigated the effects of Ga-Al-As laser irradiation on the mineralization ability of HDP cells and on Smads production as one mechanism for the transmission of laser photochemical energy to cells. HDP cells in vitro were irradiated once with a Ga-Al-As laser at 1.0 W for 500 s, and calcified nodule formation was assessed by Alizarin red S staining. The laser irradiation was greater in the laser-irradiated group than in the unirradiated control cells. Production of calcium was also increased by laser irradiation. ALP activity was higher after laser irradiation. Expression of mRNAs of Smadl, Smad7, BMPs, ALP, and osteocalcin was greater with laser irradiation. However, Smad6 mRNA expression in HDP cells was inhibited by laser irradiation. Production of BMP-2 and BMP-4 in conditioned medium was also higher after laser irradiation. These results suggest that Smads and BMPs play important roles in ALP activity and calcification upon laser irradiation of HDP cells.