Study On Expression Of Adam28 In Mouse Osteoblastic MC3T3-E1 Cells

The eruption of teeth and the remodeling of alveolar bone are normal physical phenomenon during the development of the teeth. The formation erupt pathway and remodeling of alveolar bone are both important part of the development. Osteoblasts take part in these process and play an important role. Through releasing of cytokines and responding to stimuli from exogenous and endogenous, the osteoblast and osteoclast are modified reciprocally. The two type cells are the essential of the process of bone remodeling which is normally dynamic equilibrium, such that the total amount of bone resorbed equals the amount of bone formed, maintaining the normal development of the teeth. ADAM28,a recently discovered member of a disintegrin and metalloproteinase(ADAM) family, is a secretion type glycoprotein with diverse physiological functions similarity to ADAM family such as cell proliferation, differentiation, extracellular matrix reconstruction, cleaving substrate protein, cell migration and so on. Based on research, ADAM28 genes take part in the development of tooth germ, and play an important role of regulation on the proliferation, differentiation and apoptosis of odontogenic mesenchymal cells. Meanwhile, ADAM28 genes with metalloprotease activity, is of functional importance for extracellular matrix modelling and in the ectodomain processing of molecules.Based on the mRNA and protein levels, this study is aimed to investigate the effect of adam28 on the biological characteristic of the osteoblastical cell and to analyze its possible mechanisms. Our research will provide feasible experimental and theoretical bases for further investigating the functions of ADAM28.In the study,the major contents and results were as follows:Experiment 1: cell culture, immunohistochemistry and immunofluorescence were used to examine the possible mechanisms of adam28 on the osteoblast, which demonstrated that adam28 were successfully expressed on the mice osteoblast-like cells, MC3T3-E1 in vitro. Immunohistochemistry and immunofluorescence indicated that the expression of adam28 on the MC3T3-E1 were positive. It is concluded that adam28 expressed on osteoblast-like cells (MC3T3-E1) in vitro, indicating that it may promote proliferation and differentiation of osteolast by participating in bioactivity of osteoblast.Experiment 2: cell culture and RT-PCR were used to further investigating the possible mechanisms of adam28 on mRNA levels, which demonstrated that the expression of adam28 on the were positive. It is concluded that adam28 may be synthesized by osteolast-like cell, MC3T3-E1.Experiment 3: A self-made hydrostatic compressive device was used to apply 50kPa,100kPa,150kPa on the cells respectively .After keeping on these degrees of pressure for an hour, the cells were cultured by 24h and 48h respectively. Then we observe the change of celluar shape and apply the immunochemist staining method to examine the effects of the expression of adam28 protein on the cells at different interval(24h,48h),which demonstrated that Expression of adam28 protein in mouse osteoblast-like cells(MC3T3-E1) by immunochemist staining method significantly decrease in group under strain(50kPa,100kPa,150kPa, continuously compressive pressure),but there was no difference of degree on expression between 24h and 48h group that the cells were cultured after applying CCP. It is concluded that different magnitude of continuously compressive pressure can affect expression of adam28 protein in in mouse osteoblast-like cells(MC3T3-E1) in magnitude-dependent manner, which relatively affect bone remodeling.Experiment 4: A self-made hydrostatic compressive device was used to apply 50kPa,100kPa,150kPa on the cells respectively .After keeping on these degrees of pressure for an hour, the cells were cultured by 24h and 48h respectively. Then we observe the change of celluar shape and apply RT-PCR method to examine the effects of the expression of adam28 mRNA on the cells at different interval(24h,48h),which demonstrated that Expression of adam28 on the mRNA level in mouse osteoblast-like cells(MC3T3-E1) by RT-PCR method significantly decrease in group under strain(50kPa,100kPa,150kPa, continuously compressive pressure).It is concluded that cell behavior of MC3T3-E1 is regulated by mechanical loading, and affect the procession of synthesizing adam28.