The Effects And Mechanisms Of Adrenomedullin On Human Dental Pulp Stem Cells

ObjectiveWhen the pulp is invaded by the caries progression, it can be protected through reparative dentinogenesis. Dental pulp stem cells（DPSCs） is considered to be an important source of producing reparative dentinogenesis. DPSCs shows the potential of multi-lineage differentiation, and odontoblastic differentiation plays an important role of repairing the injured pulp which is considered as the decisive factor of reparative dentinogenesis. However, current research on the proliferation and differentiation mechanisms of DPSCs are unclear. According to the previous research, high expression level of adrenomedullin（ADM） is shown on the critical time point of development during the development process of teeth. In addition, ADM shows obvious regulation effect on the osteogenesis of bone, while osteogenesis and dentinogenesis have many similarities. Based on the previous research, it is found that the expression level of ADM is significantly higher in the injured pulp than that of normal pulp by detecting the pulp tissue. And the regions of expression level mainly concentrate on the damaged regions of pulp, being around the reparative dentinogenesis. Therefore, ADM may have an effect on the proliferation and differentiation of DPSCs.The purpose of this research includes:（a） The expression level of ADM in the tissue of caries pulp, as well as the correlation of progression between ADM and caries.（b） Identification and confirmation of primary DPSCs that is subject to separation and culture in vitro, laying foundation for the further study on the regulation effect and mechanism of ADM in the proliferation, apoptosis and odontoblastic differentiation of DPSCs;（c） By adding ADM in the culture medium of DPSCs, to study on the regulation effect and mechanism of ADM in the proliferation and apoptosis of DPSCs;（d） to study the regulation effect and mechanism of ADM on the odontoblastic differentiation of DPSCs.Methods 1. Research on the correlation between caries and ADM’s expression level95 cases of caries and 100 cases of extracted healthy teeth are collected from the wisdom teeth clinically extracted in Changhai hospital（There are 44 cases of superficial caries, 32 cases of medium caries and 19 cases of deep caries according to the classification of caries）. After extractions of the teeth, the pulp tissue is immediately taken out and the expression of ADM in the pulp liquid is detected with ELISA method. The expression level and cellular localization in the pulp tissue are detected through immunohistochemistry, and the results were compared using t-test for statistical analysis.2. The culture and identification of DPSCs The healthy pulp tissue in the previous test is used for the culture. The single cell suspension is obtained by the digestion and filtration of enzyme, and conducted primary culture through limited dilution method. Expand the culture to the third generation and detect the expression levels of the markers such as DPSCs by using cell immunofluorescence STRO-1/CD146. The potential of dentinogenesis of DPSCs is determined through detecting the activity of alkaline phosphatase（ALP）, the formation of mineralized nodules and the expression level of dentin sialoprotein（DSP）.3. Research on the regulation effect and mechanism of ADM in the proliferation and apoptosis of DPSCsCultured to the third generation of primary DPSCs, cell cycle and cell apoptosis is detected through flow cytometry by adding ADM with different concentrations. The expression level of apoptosis protein and ADM on the effect on the proliferation of DPSCs was detected by Western blot. The expression level and the phosphorylation level of protein JNK/c-Jun in the critical signal pathway for regulation of cell proliferation is detected by Western blot, so as to the critical signal pathway for regulation of cell apoptosis Src/GSK-3β. In addition, the effects of ADM on the proliferation and apoptosis of DPSCs are studied by the activation of Src/GSK-3β pathway and Src/GSK-3β pathway with ADM, through co-processing for ADM and JNK/c-Jun pathway inhibitor SP600125, and ADM and Src/GSK-3β pathway inhibitor PP2.4. Research on the action and mechanism of ADM on the odontoblastic dentinogenesis of DPSCsCultured to the third generation of primary DPSCs,the expression level of relevant protein marker of odontoblastic differentiation is detected by Western blot with the treatment of ADM(10^-7mol/L)for 24 hours. Meanwhile, the expression levels of CREB, phosphorylated CREB and BMP2 are also detected. The transcription and binding site for BMP2 of CREB is detected by luciferase activity report and test Ch IP analysis. ADM promoting DPSCs’ s odontoblastic differentiation by activating CREB/BMP pathway is detected by adding H89 to inhibit CREB phosphorylation or adding Noggin to inhibit BMP pathway when treating with ADM.Results 1. Research on the correlation of caries and the expression level of ADMAccording to the results of ELISA detection: ADM level of pulp tissue fluid in the carious group is significantly higher than that in the healthy group, In addition, ADM level of pulp tissue fluid in the deep caries group is significantly higher than those in medium caries group and superficial caries group, while ADM level in medium caries group is significantly higher than that in superficial caries group. Through immunohistochemistry detection, it has been found that high expression level of ADM is shown by staining within the range of pulp near deep caries. More significant expression level is shown around the reparative dentinogenesis.2. DPSCs culture and identificationAfter 7 days, minority cells are cloned due to exuberant proliferation ability. The cells are STRO-1 and CD146 positive. After mineralization induced culture, the cells can form mineralized nodules and ALP activity is significantly increased. DSP protein of cell expression level can be shown by Western blot test.3. Research on the regulation effect and mechanism of ADM on the proliferation and apoptosis of DPSCsAfter having treated cells by 10^-7M-10-10 M ADM for 24 hr, the ratio of G2/S/M phase cells in ADM treatment group has increased significantly and multiplication time of cell is significantly lower than that of blank control group, wherein no significant differences exist in 10^-7M and 10-8M ADM treatment group. However, the promoting effect on the proliferation of DPSCs is significantly higher than other two groups. After treatment with 10-8M ADM, phosphorylation level of JNK/c-Jun protein in DPSCs has increased significantly. After joint treatment with 10-8M ADM and 10 μM SP600125, phosphorylation level of JNK/c-Jun and proliferation ability of the cells have significantly decreased.After having treated cells by 10^-7M-10-10 M ADM for 24 hours, cell apoptosis rate and expression level of Caspase3 in ADM treatment group have significantly decreased, wherein no significant differences exist in 10^-7M and 10-8M ADM treatment group. However, the inhibition effect on DPSCs’ s apoptosis is significantly higher than other two groups. After treatment with 10-8M ADM, phosphorylation level of Src /GSK-3β protein in DPSCs has increased significantly. After joint treatment for DPSCs with 10-8M ADM and 20 μM PP2, phosphorylation level of Src /GSK-3β has significantly decreased and cell apoptosis rate has significantly increased.4. Research on ADM’s effect on and mechanism of DPSCs’ s odontoblastic differentiationAfter treatment for DPSCs with 10^-7M ADM, its dyeing level of Alizarin red S is significantly higher than that in control group. The detection results of Western blot have shown that expression level of RUNX2/ALP/OCN protein in ADM treatment group is significantly higher than that in control group. In addition, expression level of p CREB in ADM treatment group has significantly increased compared to the control group. However, overall CREB level of cell isn’t significantly changed. The results of quantitative PCR and Western blot have shown that expression levels of BMP2 m RNA and protein in ADM treatment group have significantly increased compared to the control group. According to the report of luciferase activity and Ch IP results, BMP2 transcription can be promoted by CREB combined with BMP2 promoter（-1094 to-669 zone）. After joint treatment with 10^-7M ADM/5μM H89, expression level of p CREB/BMP2/RUNX2/ALP/OCN is significantly lower than the expression level obtained through single ADM treatment, while the expression level of RUNX2/ALP/OCN through joint treatment with 10^-7 M ADM/100 ng m L Noggin is significantly lower than the expression level obtained through single ADM treatment.Conclusion1. High expression level of ADM is shown on the pulp tissue of caries and positive correlation with the progress of caries.2. After separated and cultured from pulp tissue, DPSCs show the potential of odontoblastic differentiation.3. The effect of ADM on the proliferation of DPSCs by activating the JNK/c-Jun signal pathway and inhibits DPSCs apoptosis by activating Src/GSK-3β signal pathway.4. ADM promotes the odontoblastic differentiation of DPSCs by promoting CREB phosphorylation and regulating the transcription path of expression level of BMP.