The Effect Of Hey1Overexpression On Osteoclastogenesis Capacity Of Murine Monocytes RAW264.7

Tooth resorption (TR) caused by osteoclast can result in severe loss of dental hardtissue. Root canal therapy (RCT) is effective in the treatment of TR, but sometimes it isdifficult to completely terminate TR by RCT. With the further development of TR, thewhole layer of the tooth hard tissue can be perforated, leading to tooth extraction.Osteoclasts are the only cells which can cause hard tissue resorption in vivo. They alsoplay an important role in the development of tooth resorption. Osteoclasts aremultinucleated giant cells differentiated from the fusion of monocyte precursors, whichare generated in the spleen or bone marrow. Therefore, by controlling the differentiationprocess from monocytes to osteoclasts, the genesis and development of TR might beslowed down or even terminated. Notch is a classical signaling pathway, which couldregulate osteoclastogenesis, Hey1is an important effector molecule of notch signaling pathway. The role of Hey1in osteoclastogenesis has not been clearly demonstrated.. In thepresent study, by the establishment of Hey1overexpression RAW264.7cell line, theeffects of Hey1on differentiation of RAW264.7into osteoclasts were investigated, and thepossibility of inhibiting the genesis of TR by regarding Hey1as a target was evaluated.The experiment includes two parts:1. Establishment of Hey1overexpression RAW264.7cell lineAIM: To establish the Hey1overexpression RAW264.7cell line and identify it.METHODS: Amplify and extract the plasmid. Expression plasmid carrying Hey1encoding sequence was transfected into RAW264.7cells by using lipofectamine2000. Theconcentration of the Blasticidin was ascertained by preliminary experiment. Stablytransfected cells were screened by blasticidin at concentration of10μg／ml. Expressionlevels of Hey1were analyzed by RT-PCR and Agarose gel electrophoresis. RESULTS:The cells could grow well and reproduce rapidly after a long time screening with highconcentration Blasticidin. The mRNA expression levels of cell lines screened byblasticidin were significantly higher than that of normal RAW264.7. CONCLUSION:The Hey1overexpression RAW264.7cell line was established successfully.2. Observation of the effects of Hey1on differentiation of RAW264.7into osteoclastsAIM: To observe the effect of Hey1on differentiation of murine monocytesRAW264.7into osteoclasts. METHODS:50ng/ml RANKL was used to induceRAW264.7cells to differentiate into osteoclasts. The expression levels of the osteoclastmarkers including TRAP, Cathepsin and RANK were evaluated by using realtime qPCR.50ng/ml RANKL was used to induce RAW264.7cells to differentiate into osteoclasts.After5days, the numbers of osteoclasts in the normal RAW264.7cell line and in theHey1overexpression RAW264.7cell line were counted after tartrate-resistant acidphosphatase staining. Statistical analysis was performed by SPSS software. RESULTS:The expression levels of TRAP and Cathepsin K of the normal RAW264.7weresignificantly higher than those of the Hey1overexpression RAW264.7. The expressionlevels of RANK have no significant differences between the two cell lines. Cultured in thepresence of50ng/ml RANKL, the number of osteoclasts differentiated from Hey1 overexpression RAW264.7was significantly lower than that from normal RAW264.7（P＜0.05）. CONCLUSION: Hey1has an inhibitoryeffect on the differentiationof murinemonocytes RAW264.7into osteoclasts.