| Objective:The isolation and culture of human deciduous teeth pulp stem cells and dental pulp stem cells in vitro, compared to the two general biological characteristics and osteogenic induction and osteoclast differentiation. Screening and analysis of human deciduous dental pulp stem cells involved in the key factor in the process of absorption of deciduous teeth root physiological and discuss the SHED may be involved in bone remodeling of regulatory mechanisms of stem cell application in clinical orthodontic bone remodeling, and provide certain experiment and theory basis to accelerate tooth movement.Method:Purified by enzyme digestion and separation method of limited dilution to obtain human exfoliated deciduous dental pulp stem cells and dental pulp stem cells. Cell growth and morphology were observed. The growth curve was measured by MTT method. Cell clone formation rate was determined. Cell cycle was analyzed by flow cytometry. Mineralized induced liquid of shedding deciduous dental pulp stem cells and dental pulp stem cells into bone cells, respectively in the induced for 14 days, 21 days of alkaline phosphatase staining and alizarin red staining, and on the mineralized nodules were counted. By using real time fluorescence quantitative PCR detection of osteogenic induced expression of SHED and DPSCs into osteoclast related genes RUNX2, OCN, COL-1, ALP, RANKL and OPG 7d, to investigate the bone and osteoclasts ability differences between SHED and DPSCs. RT-PCR was used to detect the shed and DPSCs TNF-a, IL-6, NF-κB, OPG and RANKL expression, Western Bolot detection SHED and DPSCs whole cell protein in TNF-a and NF-κB expression levels and protein expression analysis of gray.Result:(1) SHED and DPSCs was obtained by enzyme digestion and limited dilution purification method, two kinds of stem cells were fibroblast like, and SHED showed pleomorphic, short spindle shape, flat shape, polygonal, oval and shape, DPSCs showed single cells were fusiform or star, uniform cytoplasm, round nuclei and clear nucleolus. The formation rate of DPSCs was higher than SHED, and there was a significant difference between the two stem cells (p<0.05). The growth curves of SHED and DPSCs showed "S" type SHED from the 3rd to entered the logarithmic growth phase and DPSCs from the 4d entered the logarithmic growth phase, and SHED reached peak was much higher than that of DPSCs that SHED strong proliferation ability in DPSCs. Cell cycle analysis results, G2/M+S phase, SHED was 35.54%, DPSCs was 24.14%, and the difference was statistically significant (p<0.05), also suggested that SHED has a stronger ability of cell self replication and proliferation. SHED and DPSCs in osteogenic induced for 14 days after ALP staining were positive, and most of the cytoplasm with blue purple granular precipitation, but SHED than DPSCs ALP positive expression rate was higher; osteogenic induction for 21 days after, alizarin red staining were positive, mineralized nodule formation, but SHED mineralized nodules was significantly more than that of DPSCs and SHED generated mineralized nodule area of the larger and more dense, and DPSCs generated mineralized nodules dispersed and individual nodules of the area is smaller. These results suggest that SHED has a stronger ability of osteogenic differentiation than DPSCs. (2) RT-PCR results showed that after osteogenic induction, SHED and DPSCs were expressed as bone related genes Runx2, OCN, ALP and OPG and shed of Runx2, OCN, ALP and OPG expression increased levels were higher than those in DPSCs. The results are confirmed SHED has stronger capability of osteogenic differentiation. (3) And SHED and DPSCs showed the expression of RANKL and OPG, but compared with the DPSCs SHED in osteoclast related RANKL gene expression was significantly up-regulated and reaction of osteoclast/ability of bone of the RANKL/OPG ratio was significantly higher than those of DPSCs. The results showed that compared with DPSCs, along with more osteoclasts ability. In addition, SHED and DPSCs expressed TNF-α, IL-6, NF-κB and so on inflammatory factors related to shed in TNF-a, IL-6, NF-κB expression was significantly higher than that in the control group in DPSCs. The difference is statistically significant (p<0.05); in the expression of TNF-a and SHED expression about 3 times as much as the DPSCs; in the expression of IL-6, SHED expression about 3 times as much as the DPSCs; NF-κB expression in SHED expression about 7 times as much as the DPSCs; in the RANKL/OPG ratio SHED is about 2.5 times that of DPSCs. Above differences with statistical significance (p<0.05). Bolot Western test results showed that the expression level of TNF-a and NF-κB was significantly higher than that in SHED(p<0.05).Conclusion:Success from deciduous teeth and permanent teeth with pulpitis and isolated SHED and DPSCs. Compared with DPSCs, SHED has strong proliferation ability, doubling time is short, and has higher clone forming ability. After osteogenic induction, SHED has a stronger ability of osteogenic differentiation than DPSCs. In addition, SHED also has a certain ability to break bone. The primary physiological root resorption and bone remodeling in the process of SHED may through the secretion of inflammatory cytokines such as TNF-a induced activation of NF-κB signaling pathway and NF-κB signaling pathway activation in RANK/RANKL/ OPG system regulation, RANKL/OPG ratio was significantly larger, promote osteoclast differentiation and maturation, and root bone remodeling around, suggesting that SHED may be involved in physiological root resorption of bone remodeling, for stem cells used in orthodontic clinic bone reconstruction, accelerate the orthodontic tooth movement to provide certain experimental and theoretical basis. |
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