| object This study aimed to investigate the effect of inflammatory stimuliation on dental pulp cells(DPCs)and human umbilical cord mesenchymal stem cells(hUCMSCs)by assessing their proliferation and expression of mRNA including Interleukin 6(IL-6),Interleukin 10(IL-10)and hepatocyte growth factor(HGF)in lipopolysaccharide(LPS)of different concentrations induced microenvironment.The coculture system of hDPCs and hUCMSCs was established to compare the mRNA expression and protein levels of cytokine in each group to explore the regulation effect of the coculture system on cells.Methods(1)HUCMSCs and hDPCs were cultured for different periods(6,12,24,and 36 h)stimulating with LPS(1,10,20 and 50?g / mL)for 6,12,24 and 36 hours.Proliferation was assessed with MTT and mRNA levels of IL-6,IL-10 and HGF were detected by QRT-PCR.(2)DPCs,UCMSCs and co-culture system were cultured with LPS at a concentration of 10 ?g/ml.Cell proliferation was detected by MTT assay,the levels of mRNA and secretion of IL-6,IL-10 and HGF were measured by QRT-PCR and enzyme linked immunosorbent assay(ELISA)at 6,12,24 and 36 hours.Results(1)Effect of LPS on proliferation and mRNA expression of DPCs and UCMSCs:(1)The proliferation of DPCs was significantly enhanced at a concentration of 10 ?g/mL LPS,while decreased at concentrations of 20 and 50 ?g/mL LPS(P<0.05).There was no significant difference in the proliferation of UCMSCs cultured with LPS of different concentrations(P>0.05).(2)It was observed that mRNA expressions of IL-6,IL-10 and HGF showed statistical increase when cells cultured with LPS at all concentrations except the concentration of 1?g/mL(P<0.05).(2)Effect of LPS on proliferation and expression of inflammatory cytokines of cells in direct coculture system of hUCMSCs and DPCs:(1)The proliferation of cells in coculture system was significantly enhanced compared with the DPCs cultured with LPS at 6,12 and 36 hours(P<0.05),while similar to UCMSCs cultured with LPS.(2)The mRNA expression of IL-6 in coculture system was statistically decreased compared with DPCs cultured with LPS,while enhanced compared with UCMSCs cultured with LPS(P<0.05).(3)The mRNA expression of IL-10 and HGF in coculture system was markedly enhanced compared with DPCs cultured with LPS at 12,24 and 36 hours,while decreased compared with UCMSCs cultured with LPS(P<0.05).(4)The mRNA levels of IL-6/IL-10 and IL-6/HGF in coculture system were significantly lower than DPCs cultured with LPS(P<0.01);(5)There was significantly high release of IL-6 in coculture system and DPCs cultured with LPS compared to DPCs cultured without LPS(P<0.01).The secretion of IL-6 in coculture system was makedly decreased compared to DPCs cultured with LPS at 36 hours(P<0.01).(6)The secretion of of IL-10 and HGF in DPCs cultured with LPS statistically increased compared to DPCs cultured without LPS(P<0.05),while the release of IL-10 and HGF in coculture system was lower than DPCs cultured with LPS(P<0.01).(7)The protein levels of IL-6/IL-10 and IL-6/HGF in DPCs cultured with LPS were significantly higher than DPCs cultured without LPS(P<0.01),while those in coculture system were significantly lower than DPCs cultured with LPS(P <0.01),there was no significant difference between coculture system with DPCs cultured without LPS in a certain period of time(P> 0.05).Conclusion(1)The concentration of 10?g/mL LPS is capable of promoting DPCs proliferation and mRNA expression of IL-6,IL-10 and HGF,we choose 10 ?g/mL LPS for future research through comprehensive consideration.(2)The coculture system of UCMSCs and DPCs can promote proliferation,significantly decrease the expression of pro-inflammatory cytokine IL-6,suppress the expressions of anti-inflammatory cytokines IL-10,HGF,elevate levels of IL-6/IL-10 and IL-6/HGF. |
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