The Study On The Function Of STAT3 In Differentiation Of Human Dental Plup Cells Induced By Connective Tissue Growth Factor

Pulp damage caused by inflammation,traumatic is increasing year by year.At present we have not found therapies that could completely recover the function of pulp tissue. Previous research had confirmed that Human Dental Plup Cells(h DPCs)have a natural proliferative and differentiated potential. In fact, cultured dental pulp cells have an ability to form calcified tissue that is regulated by a complex sequence of cytokines in vitro. This suggests that cytokines induce regeneration of the injured dentin-pulp complex. Connective tissue growth factor(CTGF)can promote fibrosis intensively,can enhance the mineralization of dental pulp cells, but it does not stimulate the proliferation of human dental pulp cells. But there is rare reports to its signal transduction mechanism.In recent years,we found that Janus protein tyrosine kinase-Signal transducer and activator of transcription( JAK-STAT) is ssociated with inflammation,cell proliferation and differentiation etc. The study will discuss that whether the STAT3 in differentiation of human dental plup cells induced by connective tissue growth factor is working.Part I The effects of CTGF on the proliferation and differentiation of human dental pulp cellsObjectives:To isolate and cultivate human dental pulps cells in vitro and to observe the effect of CTGF on proliferation and differentiation of the cells.Methods:Pulp samples were obtained from healthy, complete third molars or premolar for impacted or orthodontic reason(with the patients’ infromed consent).The pulp tissue was removed under the sterile conditions,cultured according to the tissue explant-collagenase digestion method and be identified in vitro. CTGF of different concentration(0ng/ml,10ng/ml,20ng/ml,50ng/ml) was used to treat h DPCs for different long time and its effects on h DPCs proliferation and the formation of mineralized nodules were detected by MTT and Alizarin red staining.Results:It can be successfully cultivated fibroblast-like cells through the method of tissue explant-collagenase digestion,the cells observed by Inverted Microscope are found to be primarily typical long-shuttled appearance, well-rounded cell body, uniform cytoplasm, round or oval nucleus in the middle of cell, and clear nucleolus. Immunocytochemistry staining shows that h DPCs are positive for vimentin and negative for cytokeratin.CTGF of the three concentrations(10ng/ml,20ng/ml,50ng/ml) have no toxicity on cells and can promote h DPCs proliferation on the third day(P<0.05),increasing with the extension of time;Alizarin red results show that the mineralization nodules of CTGF group is obviously higher than that of control group.Conclusions:h DPCs were isolated and cultured successfully by the method of tissue explant-collagenase digestion. CTGF can promote the proliferation and differentiation of h DPCs in a certain concentration. Part II The effects of CTGF on protein expression of STAT3 in human dental pulp cellsObjectives:To observe the protein expression of STAT3 in h DPCs treated with CTGF alone.Methods:Generation 4 h DPCs in logarithmic phase were trypsinized and made into cell suspension. After climbing to the carry sheet glass,cells were randomly divided into CTGF treated groups,control group and Stattic(The STAT3 specific inhibitor) +CTGF treated groups. Immunocytochemistry staining was performed to detect the expression of STAT3 active form(phosphorylated protein) respectively.Results:As h DPCs treated with CTGF for 30 minutes,it can be saw that brown granules are mainly distributed in the nucleus, indicating that Phos-STAT3 is expressed in the nucleus primarily;while control group is the very opposite of the experimental group, brown granules are mainly distributed in the cytoplasm,showing that Phos-STAT3 is expressed in the cytoplasm primarily. However,after having been treated with inhibitors of STAT3, brown granules are mainly distributed in the cytoplasm less than the control group, little is found in the nucleus.Conclusions:CTGF can increase expression of Phos-STAT3 protein obviously,and promote the Phos-STAT3 expressed in the nucleus. STAT3 inhibitor can inhibit phosphorylation of STAT3 specially. Part III The role of STAT3 in CTGF-induced differentiation of human dental pulp cllsObjectives:To observe the role of CTGF in h DPCs differentiation by detecting mineralized nodules and m RNA expression of ALP, DMP-1, OCN and OPN after cells have been exposed to CTGF with or without inhibitors.Methods:Generation 4 h DPCs in logarithmic phase were made into cell suspension. Then the cells were subcultured in 6-well plates with 104cells/ml,When the cells were confluent,cells were randomly divided into blank control group, masculine group, and experiment group,in which the cells were treated with Stattic for 30 minutes first. After 7 days,14 days,28 days,the formation of mineralized nodules and m RNA expressions of ALP, DMP-1, OCN and OPN were detected by Alizarin red staining and Real-time PCR.Results:Alizarin red results show that the mineralization nodules is obviously decreased after treated with Stattic, compared with positive control group.After 7 days, compared with control group,m RNA expression of ALP,OCN, OPN, DMP-1 in h DPCs decreased obviously after treated with Stattic,while the expression of CTGF group is increased(P<0.05). At 14 days, m RNA expression of ALP decreased after treated with Stattic, and the expression of CTGF group is increased with statistical significance(P<0.05). MRNA expression of OCN,OPN revealed no difference with control group(P>0.05). MRNA expression of OCN decreased and m RNA expression of OPN increased after treated with CTGF(P<0.05). MRNA expression of DMP-1 in h DPCs decreased of all groups with statistical significance(P<0.05).Conclusions STAT3 plays an important role in the process of CTGF-induced h DPCs differentiation.Above all,it is indicated that CTGF could promote proliferation and differentiation of human dental pulp cells and activate STAT3,which increased phosphorylation protein expression of STAT3 in h DPCs,and switch to the nucleus.The results provide new experimental evidence for molecular mechanism study of CTGF-induced h DPCs differentiation, and for potential new methods of pulpitis therapy in clinical.