Effect Of T3 On Odontogenic/Osteogenic Differentiation Of Periodontal Ligament Stem Cells And Its Mechanism

Thyroid hormones secreted from thyroid gland,includes the active hormone triiodothyronine(T3)and its prohormone thyroxine(T4).T3 is the true hormone,which could be converted from T4 and its effects on target tissues are roughly fifteen times more potent than those of T4.It is well known that T3 are important regulators of bone development and metabolism,and they could influence osteoblastic differentiation.It has been reported that depletion of the receptors for thyroid hormones,mimicking hypothyroidism,could result in severe distortions of the growth plate and delayed bone development.Many studies in primary cultured or immortalized osteoblastic cells have demonstrated that T3 could have a positive effect on osteogenesis.However,the effect of T3 on human periodontal ligament stem cells has not been studied yet.Part I.Isolation and culture of human periodontal ligament stem cells and effects of T3 on proliferation and cell cycle of PDLSCsObjective: To obtain PDLSCs and make clear the effect of T3 on proliferation and cell cycle of PDLSCs cells.Methods: PDLSCs were isolated and cultured by enzyme digestion method.Cell counting kit-8(CCK-8)assay was performed to evaluate the proliferation activity of T3-induced PDLSCs.The cell cycle was examined by flow cytometry.Results: PDLSCs were isolated and cultured.As compared with the control group,T3 almost had no influence on proliferation of h PDLSCs(P <0.05).Flow cytometry showed the cell cycle of PDLSCs treated with T3 was not significant different(P <0.05)with the control group.Conclusions: T3 almost had no influence on proliferation of h PDLSCs.There was no significant difference between T3 and control group in terms of cell cycle.Part II.Effects of T3 on differentiation of PDLSCsObjective: To observe the effects of T3 on the odontogenic/osteogenic differentiation of PDLSCs.Methods: Odonto/osteoblastic proteins(COL-1、ALP、RUNX2、DSP、OSX、OPN、OCN)and genes(COL-1、ALP、RUNX2、DSPP、OSX、OCN)in PDLSCs were respectively assessed by western blot and real-time PCR(RT-PCR)technique.Alizarin red staining was to detect the formation of mineralized nodules after 14 days and cetylpyridinium chloride(CPC)assay was to detect the calcium concentration.Results: The protein expression of COL-1、ALP、RUNX2、DSP、OSX、OPN、OCN and gene expression of COL-1、ALP、RUNX2、DSPP、OSX、OCN were significantly higher than those in control group in PDLSCs in vitro.Alizarin red staining showed that more mineralized nodules were formed in PDLSCs treated with T3 and a significant higher calcium concentration was detected in this group.Conclusions: T3 can promote the differentiation of PDLSCs into odontogenic/osteogenic lineages in vitro.Part III.NF-κB signal pathway mechanisms embeded in the differentiation of PDLSCs treated with T3Objective: To explore the NF-κB signal pathway mechanisms during the differentiation of PDLSCs induced by T3.Methods: The status of NF-κB signal pathway was detected by western blot according to the expression of several key proteins(P65,IκB-α,phosphorylation P65 and phosphorylation IκB-α).BMS345541 inhibited the NF-κB pathway and western blot was to check the related proteins.Alizarin red staining inhibited by BMS345541 was used to evaluate the formation of mineralized nodules and the calcium concentration was determined by CPC assay after 14 days.Results: P65 was observed to transfer from cytoplasm to nuclei,as indicated by western blot p-P65 were rapidly upregulated at 15 min.IκB-α degraded rapidly in cytoplasm,as as indicated by western blot IκB-α were rapidly downregulated at 30 min.These findings suggest that NF-κB pathway was activated during the treatment of T3.Nuclear translocation of P65 was decreased by BMS345541-treated as indicated by the expression of p-P65 in cytoplasm were significantly reduced and IκB-α were significantly increased.The formation of mineralized nodules was obviously reduced treated with BMS345541 and the calcium concentration was also reduced significantly.Conclusions: NF-κB signal pathway was activated instantly when PDLSCs were treated with T3.Therefore,we conclude that T3 can promote odonto/osteoblastic differentiation of PDLSCs via NF-κB signal pathways.