Study On Epithelial Proliferation And Cyst Fluid Microvesicles In Odontogenic Keratocysts

Part 1.Overexpression of Fra-1,c-Jun and c-Fos in odontogenic keratocysts:potential correlation with the proliferative characteristicsObjective: The purpose of this study was to explore the potential involvement of Fra-1,c-Jun and c-Fos,three vital members of the AP-1 complex,in the pathogenesis of odontogenic keratocysts(OKCs).Methods: Tissue samples,containing 10 normal oral mucosa(OM),10 dentigerous cysts(DC)and 32 OKC specimens,were applied to investigate the expression levels of Fra-1,c-Jun and c-Fos by immunohistochemistry.The association between Fra-1,c-Jun and c-Fos expression levels and markers of proliferation [Ki-67,proliferating cell nuclear antigen(PCNA)],anti-apoptosis(Bcl-2)was then investigated in the OKC serial tissue sections by double-labelling immunofluorescence analysis and hierarchical analysis.Results: The results showed that Fra-1,c-Jun and c-Fos expression levels were increased significantly in OKCs compared to these in OM and DC tissue samples.Meanwhile,the expression levels of Fra-1,c-Jun and c-Fos were associated positively with the expression levels of Ki-67,PCNA and Bcl-2,as confirmed by double-labelling immunofluorescence analysis and hierarchical analysis.Conclusions: This study revealed for the first time that Fra-1,c-Jun and c-Fos were overexpressed in OKCs and had a close correlation with proliferation and anti-apoptosis potential of OKCs.Part 2.Characterization of cyst fluids microvesicles in patients with odontogenic keratocystsObjective: The aim of this study was to examine the level and basic characteristics of cell-derived microvesicles(MVs)in cyst fluids of odontogenic cysts.Methods: For this purpose,MVs from the cyst fluids(CFMVs)of odontogenic cysts were purifed by a classic differential centrifugation method and characterized by a transmission electron microscope and fluorescence microscope.Flow cytometric analysis was used to determine the size,concentration and cellular origins of the CFMVs.Moreover,the expression level of receptor activator for nuclear factor-?B ligand in the OKCs was evaluated by immunohistochemical staining and then analyzed for its correlation with the concentration of CFMVs.In addition,reverse transcription?quantitative polymerase chain reaction(RT?q PCR)and tartaric?resistant acid phosphatase(TRAP)staining were performed to examine the osteoclastogenesis of mouse bone marrow?derived macrophages(BMMs)in response to CFMVs.Results: The results revealed that the levels of total CFMVs were signifcantly elevated in OKCs compared with dentigerous cysts(DCs)and radicular cysts(RCs).In addition,in vitro experiments further revealed that CFMVs derived from OKCs patients could be taken up by BMMs,leading to a significant increase in the m RNA expression levels of nuclear factor of activated T?cells 1(NFATc1)and TRAP.Moreover,TRAP?positive multinucleated osteoclasts were successfully cultured in the presence of macrophage colony?stimulating factor(M?CSF)and CFMVs with BMMs.Conclusions: Our findings indicate that patients with OKCs have higher levels of CFMVs compared with patients with DCs and RCs,which may be associated with the bone resorption of OKCs.Part 3.Study on the cyst fluid lymphocyte?derived microvesicles of odontogenic keratocystsObjective: Leukocyte-derived microvesicles(LMVs)include neutrophil-,lymphocyte-and monocyte-derived MVs.LMVs act as proinflammatory mediators in autoimmune diseases,infectious diseases and vascular diseases.The present study examined the hypothesis that the percentage of LMVs was increased in patients with inflamed odontogenic keratocysts(OKCs),and investigated the biological effects of Jurkat cell?derived MVs on the fibroblasts of OKCs in vitro.Methods: Cyst fluid MVs,obtained by centrifugation of samples from 20 patients with inflamed OKCs,3 patients with uninflamed OKCs,15 patients with radicular cysts(RCs)and 12 patients with inflamed dentigerous cysts(DCs),were analyzed by transmission electron microscopy,dynamic light scattering and immunofluorescence staining.The percentages and concentrations of cyst fluid LMVs were further determined by flow cytometry.The cytokine levels of apoptotic Jurkat cell-derived MVs and Jurkat cell supernatants were compared by cytokine antibody arrays.Fibroblasts were isolated from 3 patients with OKC and co-cultured with apoptotic Jurkat cell-derived MVs with or without interleukin(IL)-15R? to detect the levels of matrix metallopeptidase 9(MMP-9)and receptor activator of nuclear factor-?B ligand(Raza et al.)by reverse transcription-quantitative polymerase chain reaction and enzyme-linked immunosorbent assay.The supernatant from Jurkat MVs?treated fibroblasts was collected to make conditioned medium in which the osteoclastogenesis of Raw264.7 cells was determined.Antibodies against human soluble(s)RANKL were added to the conditioned medium to investigate the inhibitory effects.Results: Mean percentages of lymphocyte? and neutrophil?derived MVs were significantly higher in inflamed OKCs than in DCs.Significant elevations in IL-15 were detected in apoptotic Jurkat cell-derived MVs compared with that in Jurkat cell supernatant.Furthermore,higher levels of MMP-9 and RANKL were determined in Jurkat cell MVs?treated OKC fibroblasts,and this was partially blocked by IL-15R?.Increased osteoclast-like cell formation was observed in the Jurkat MVs?treated fibroblasts supernatant and Raw264.7 co-culture groups.The anti-human s RANKL antibody in the Jurkat MVs?treated fibroblasts supernatant group decreased the osteoclastogenesis of the Raw264.7 cells.Conclusions: These results indicate that LMVs serve as novel communication tools that contribute toward the bone resorption of inflamed OKCs by inducing RANKL of OKC fibroblasts via IL-15.