Investgate The MiRNAs Profile Of The Human Dental Pulp Cells (DPCs) Being Induced Into Odonblast Differentiation By BMP-2

It has been found that the Bone Morphogenetic Protein - 2 (BMP-2) plays an important role in the process of the dental pulp cells (DPCs) into odontoblast differentiation. Numerous studies tell that, the BMP's direct induction or transfection by genes can led to the odontoblast differentiation of the DPCs.Over the past decade, we found that MicroRNAs are a class of small RNA with regulatory functions in 30%--90% of human genes. This kind of small fragments noncoding RNA plays an important role in gene regulation. MiRNAs can control the cells' differentiation, proliferation, apoptosis and a variety of important biological actives. However, the biological mechanism of miRNAs involved in the process of BMP-2's odontoblast differentiation in DPCs have not been future studied. The purpose of this study is supposed to select the MicroRNAs in the process of BMP - 2 induced dental pulp cells.Aim:1. Isolate and culture the human dental pulp cells ( hDPCs )in vitro.2. Investigate the optimum condition of the bone morphogenetic protein-2(BMP-2) on the biologic activity of Human dental pulp cell (DPCs) in vitro.3. Investigate the differentiation markers of miRNAs in the human dental pulp cells when induce it into odontoblast by BMP-2. Prepare for the further studies in the biological mechanism of miRNA acts on the BMP-2's induction of dental pulp cell differentiation.Methods:1. Isolate and culture the human dental pulp cells (DPCs) in vitro. Collect the 4-6 generations cells for vimentin, cytokeratin immunohistochemistry to identify cell sources.2.use the BMP-2 with different concentration (50?100?150ng/ml) to induce the stable passage of dental pulp cells ( passage 4 to 8 ). The expression of ALP ?DMP-1 and DSPP are quantified by RT-PCR to investigate the optimum condition of the BMP-23. Differences of the miRNAs between the experimental group and the control group is testified by the miRNA microarray. Then select some different expressed miRNAs using bio-information software. The correctness of the miRNAs profile is been varied by the RT-PCR.Results:1.The Dental pulp cells were spindle-shaped with plenty of plasma. Passaged dental pulp cells showed a strong ability to grow in vitro. Immunohistochemical staining suggested that the cells come from the mesoderm with the positive expression of Vimentin and the negative expression of cytokeratin.2.The best concentration of the BMP-2 turned to be 100ng/ml with the induction of the dental pulp cells with different concentration (50?100?150ng/ml. The result was proved by the RT-PCR.3. using the miRNA microarray, we found that the induced group and the control group have significant differences in the miRNA expression profiling. A plurality of miRNAs up-regulated or down-regulated. In the profile, there are about 52 RNAs change, about 23 up-regulated and 29 down-regulated . The results of RT-PCR was consistent with the microarray result in the different expression between the two groups.Conclusion:1. The dental pulp cells showed a strong ability to grow in vitro. It provides the basis for the further study of this cells.2. When Dental pulp cells (DPCs) differentiate into odontoblasts, BMP -2 plays a key role in this process, accompany with a plurality of miRNAs up-regulated or down-regulated. The result of this experiment can help us to make a further research in the biological mechanism of miRNA in our body. Also,this provides a new thought of the human dental pulp cells' treatment and regeneration.