Study On Expression Of Cofilin And Osteogenesis Related Gene Of Rho/rock Pathway In Osteoblast-like Cell After Stress Loading

Skeletal changes with the environment and external stimuli, for example the mechanical stress stimulation. In clinical work, the use of bone remodeling have very significant results, for example, the absorption and reconstruction of orthodontic tooth movement in the alveolar bone; bone growth by certain distraction stimulates in distraction osteogenesis and in the fracture healing process. Certain stress stimuli can promote new bone formation, thus healing. The mechanical signal activates the effect on osteoblast function, and then be translated into biochemical signals, finally to promote the morphological changes of bone and function adjustment.Complex mechanical signal process in osteoblast has been confirmed that cell cytoskeleton is the key mechanical sensing components. Cell cytoskeleton, fiber structure of the cell, consists of microtubules, intermediate filaments and actin filament. These networks are considered to be the foundation to maintain constant tension, local small deformation will affect the overall framework of tension, and then activate cell reaction. ADF/Cofilin play an important role in controlling the dynamic reorganization process of actin filaments. ADF /Cofilin family protein preferentially binds to ADP in the position of the tip of the actin filaments, and enhance depolymerization of F- actin through cutting F-decomposition. LIMK1(LIM kinase) phosphorylating Cofilin inhibits depolymerization level of Cofiln. Rho protein is in the upper reaches and moderate cytoskeleton. In conclusion, Rho protein and Cofilin plays a unique role in cytoskeletal signal process.Osteosarcoma cell MG-63 was applied in this experiment. RT-PCR, Western-blot and other methods was used to analyze the expression of Rho protein, downstream effector molecules ROCK and Cofilin, thus throwing light to the process of signal transduction in cells. We also inhibit Rho A to observe the effect of cell signal pathway on osteogenesis.Objective: this experiment using Flexcell mechanical loading device, mechanical stimulation on osteoblast like cells, detection gene and protein expression changes of Rho, ROCK and Cofilin. After blocking the Rho/ROCK pathway, the expression of Cofilin protein was observed and the effect on the osteogenic effect with or without mechanical loading.Methods: 1) 12% tension loading was applied on osteoblast like cells, respectively, 1 hours, 4 hours, 8 hours, 12 hours, 24 hours. We used RT-PCR, Western-blot to detect the expression of Cofilin. 2) 12% tension loading was applied on osteoblast like cells, respectively, 1 hours, 4 hours, 8 hours, 12 hours, 24 hours. We used RT-PCR to detect Rho, ROCK gene expression, Western-blot was used to detect the expression of protein. 3) We used the cells were treated with Rho/ROCK signal pathway inhibitor. After the pretreatment, 12% tension loading was applied on osteoblast like cells, respectively, 1 hours, 4 hours, 8 hours, 12 hours, 24 hours. We used RT-PCR to detect the expression of ROCK and Cofilin. Western-blot was used to detect the expression of ROCK and Cofilin protein after 8 hours tension loading. 4) We used the cells were treated with Rho/ROCK signal pathway inhibitor. After the pretreatment, 12% tension loading was applied on osteoblast like cells, respectively, 1 hours, 4 hours, 8 hours, 12 hours, 24 hours. RT-PCR was used to detect the expression of ALP, OCN and Runx-2 which were downstream gene in signal pathway on osteogenesis, with and without pretreatment of inhibitor.Results: After tension loading, expression level of Cofilin increased to the highest in 8 hour group, and then decreased. Rho, gene expression of ROCK began to increased in 4 hours group, reaching a peak at 8 h, and then decreased. After pretreatment of inhibitors which inhibited Rho/ROCK signal pathway, the expression of Cofilin decreased compared with group without inhibition, but still higher than the control group, which means that Blocking Rho, ROCK pathway could weaken the effect of tension loading on expression of osteogenesis effect genes.Conclusion: Rho and downstream effector molecules ROCK have a certain role in the regulation of Cofilin after tension loading, and impact the osteogenesis of osteoblasts.