| Human dental pulp stem cells(hDPSCs) is undifferentiated cells found in dental pulp tissue. It can update itself, get the odontogenic differentiation potentiality under the certain condition of induction differentiation, and maintain the stability of the organization.Compared with other adult stem cells, human dental stem cell showed a strong vitality of proliferation, and differentiation; compared with BMMSCs, hDPSCs may be easier odontogenic differentiation. When cells induced by a high β-tubulin III(TUBB3) and MAP – 2 level in vitro, it seems to be certificated of having a strong neural differentiation potential. This differentiation vitality of cells is inseparable with the inflammatory microenvironment. ALP, OCN and RUNX- 2 relatived genes increase obviously and have stronger self-replicating force in those dental pulp stem cells under inflammatory stimulirather than normal hDPSCs. So we think hDPSCs, as a kind of ectomesenchymal stem cells, may participate in reparative dentine formation, but its regulatory mechanism is not clear.Both mitogen-activated protein kinase(MAPK) and nuclear factor-kappa B(NF-κB)are important cell signaling pathway in a variety of pathophysiological mechanism.MAPK is an important regulator of bone defect model, influences the absorption and regeneration of bone cells, and promotes deposition and mineralization of bone matrix.Existing research shows that many kinds of drugs can affect development of human mesenchymal stem cells through p-JNK, and p-p38 pathway; in hDPSCs, some drugs promote cell differentiation through MAPK P38 and ERK pathway but some are not. Thus,in different intervention condition and cell types, there are differences in the way they regulate differentiation.Aspirin is a kind of nonsteroidal anti-inflammatory drugs, having stable curative effect, less side effects and wide treatment range. With the deepening of the clinical application research of aspirin, some new pharmacological activity has been found.Aspirin has a variety of biological pathways, in down regulating the level of inflammatory cytokines. It also play an important role in regulation of cell differentiation: acetylsalicylic acid can suppress bone marrow stromal cells proliferation, differentiation and migration.Aspirin is commonly used as anti-inflammatory drugs, while its effects on hDPSCs is unclear. Whether aspirin can adjust hDPSCs differentiation, has not been reported. This experimental study of aspirin used to stimulate cell proliferation and differentiation, and differentiation of related mechanisms, to infer hDPSCs in the body of the state, provide experimental support for clinical medication. The principle results are as follows:1. The isolation, culture and identification of h DPSCsCollect 18 to 25 healthy adults’ orthodontic tooth or third molars without caries,combine improved tissue block and enzymatic digestion method, and successfully cultivate the original generation of human dental pulp cells(human dental pulp cells,hDPCs). Limited dilution method is used to pick out the growth state of monoclonal celland expand culture get good purification hDPSCs, using flow cytometry instrument to mark the cell surface antigen CD105 and CD146 to identificate. Using mineralized induced experiment tests their osteogenesis differentiation ability, suggesting that the cells originate mesenchymal tissue and can be induced by osteogenesis differentiation.2. The influence of Aspirin on hDPSCs proliferation in vitroDetermination of MTT(dimethyl thiazolyl diphenyl tetrazolium) results showed that the influence of different concentration of aspirin for hDPSCs has the characteristic of dose dependent. Based on the pharmacokinetics of aspirin, we choose different concentrations of aspirin(5, 0.5, 0.05, 0.005) stimulation on hDPSCs for 1 day, three days,five days, seven days, respectively. When the concentration of aspirin for 0.05 mmol/L and 0.05 mmol/L after three days, the trend of cell proliferation is significantly increased(P < 0.05), the concentration of 0.5mmol/L for promoting effect is not obviously compared with the control group. When the concentration is greater than 5mmol/L, aspirin significantly inhibits the growth of cells(P < 0.05), and thus we speculated that high concentration of aspirin on hDPSCs may have toxic effects. And cell proliferation of Edu experiment are consistent with the results determined by MTT.3. The influence of Aspirin on hDPSCs differentiation in vitroDifferent densities(0.005, 0.05, 0.5) of aspirin were added into the stimulation mixture fluid; mineralized group and blank group were control. After 14 days, alizarin red staining method was used to observe mineralized nodules of each group and PCR technique is to detect the contents of the corresponding the expression of osteogenic related genes. Alizarin red results show: compared with control group, the concentration of0.5mmol/L and 0.05mmol/L aspirin can reduce the number of mineralized nodules, while there exists no obvious difference between this two groups anyway, but 0.005mmol/L aspirin group shows no significant difference with the control. Observed under inverted microscope, the cells are in good condition, and mineralized nodules are homogenous.Alizarin red quantitative result shows no difference with the observation. PCR result shows that compared with control group, of ALP, OCN, DMP1 gene have different degreeof decline in 0.5 mmol/L and 0.05mmol/L aspirin mineralization groups, which the most obvious tendency is 0.5mmol/ L stimulation group. Thus we speculate that aspirin may have inhibition effects on hDPSCs mineralization differentiation.4. The involving signaling pathway of Aspirin on differentiation of hDPSCsP4 hDPSCs were added 0.5mmol/L aspirin, and induction time was 15 min, 30 min,60min, and add the appropriate pathway inhibitor in 60 min, with 10% ɑ-MEM as a control group, the relevant pathway inhibitors including MAPK(ERK, JNK and P38)inhibitor(U0126, SP600125 and SB203580), NF-κB inhibitor of P65 PDTC. Total protein was extracted using Western blot technique, to explore the role of mitogen-activated protein kinase(MAPK) and nuclear factor-κB(NF-κB) pathway in hDPSCs mineralization of aspirin. Compared with the control group, 0.5mmol / L aspirin inhibits the formation of mineralized nodules; western blot showed that irritation with prolonged time, expression levels of p-JNK and p-p38 was significantly decreased, while there was no significant change of p-ERK. The expression of p-p65(NF-κB) was increased.Mineralization induceing 14 days, corresponding pathway inhibitor with aspirin group and mineralization showed as control groups, the gross appearance and microscopic results of Alizarin Red, were consistent with the results of western.According to the above results we speculate that aspirin inhibit the dental pulp stem cells differentiation possibly by inhibiting the MAPK p38, JNK signaling pathway, and promoting NF-κB pathway, while ERK pathway may not participate.Further study the role of aspirin on cell differentiation about other signaling pathways taking part in, will help to investigate the effect of dental pulp stem cells clinical applications of this drug as well as be conducive to further explore its application in pulp regeneration area. |
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