Odontoblast Differentiation-Related MicroRNAs Expression In Human Dental Pulp Cells

Backgroud MicroRNAs (miRNAs) are 20~22 nt noncoding RNAs that is incorporated into the RNA-induced silencing complex (RISC) where miRNA is degraded, with miRNA serving as a guide for its mRNA target. miRNA-armed RISC can enforce either degradation of mRNA or inhibition of mRNA translation to suppress the expression of protein. As internal RNA of organism and molecular which can tweak the gene expression by regulate the expression of genes involved in many biological processes,including development, differentiation and apoptosis. In recently, many evidence indicates that miRNAs play essential roles in embryogenesis, cell differentiation and pathogenesis of multiple cells by negative regulation of key gene.Pulp tissue plays a very important role in nutrition metabolism,feeling,repair and other physiological functions. Through studies of pulp help us know more about the differentation of HDPCs, pulp calcification, dentinogenesis and some the relevant regulation factors. The HDPCs can be induced and differentiated into odontoblast and form the dentin type structure, which indicated its prospect of being the seed cell for the dentin regenerates.Objectives The purpose of this study is to by determin the expression profile of the miRNA in HDPCs and odontoblas induced from HDPCs, to screen its differential expressing miRNA before and after induced differentation. And to forcast its target gene, finds sequence-specific miRNA of HDPCs. To verified, it may lay the groundwork for discussing the regulatory effect of the miRNAs for the biological function of HDPCs.Methods (1) HDPCs were isolated and cultured by enzyme digestion and tissue patch, the Morphological feature of HDPCs primary and passage cultered were observed, surface molecule marker was determined with immunocytochemistry, the HDPCs were identified.(2)Induced and differentiated HDPCs into osteoblasts with dexamethasone,β-glicerophosplale, vitamin C.(3) The expression profile of HDPCs and odontoblast induced from HDPCs were determined by miRNA gene chip technique, analysis and screening the differential expressing miRNA during the differentiated process. And to forcast its target gene by miRGen data base.(4) The differential miRNAs of HDPCs and odontoblas induced from HDPCs which were screened by miRNA gene chip technique were identified by RT-PCR method.Result (1)Cells were isolated and cultured by enzyme digestion and tissue patch, cells phenotype were examined by immunohistochemistry: vimentin and typeⅠcollagen are positive.(2)HDPCs can be induced and differentiated into osteoblasts in the culture medium with the dexamethasone,β-glicerophosplale, vitamin C, formed calcified nodule, the ALP increased, calcified nodule Von Kossa were positive expression.(3) The expression profile of HDPCs and odontoblas induced from HDPCs were determined by miRNA gene chip technique, the differential expressing miRNA were screened, Compared with the HDPCs which cultured in the ordinary culture medium,there were 72 kinds of miRNA expressing 1.5-fold up-regulated and 61 kinds of miRNA expressing 1.5-fold down-regulated in the mineralization induction culture medium.(4)By analyze the differential expressed miRNA with miRNA target forecast tools which find 35 miRNAs with target gene,the total of target genes of 35 miRNAs is 1327, the total of relations of miRNA and target gene is 2158, the result which hsa-miR-150 was identified by RT-PCR is accordant with chip test result.Conclusion (1) Human dental pulp cells which can be insolated from dental pulp tissue by enzyme digestion and be induced to osteoblasts in vitro.(2)There are plenty of miRNAs in HDPCs and odontoblats induced from HDPCs.(3) Between HDPCs and odontoblasts which induced from HDPCs exist some differential expressing miRNAs which may play essential roles of regulation in the process of HDPCs differentiated into odontoblast.