An Study On The Role Of Cofilin On The Osteoblast Mechanical Signal Transduction

Normal bone is in a dynamic equilibrium state of absorption and reconstruction.Mechanical stimulation is one of the necessary conditions of maintaining the balance. Agrowing number of studies confirm that tensile stress can effectively promote the activityand function of bone. Osteoblasts can perceive both in vivo and in vitro mechanicalstimulation through various channels, and transform into biochemical signals that mediaterelevant sensitive gene expression, the synthesis of various enzymes such as activesubstances, which in turn activates the signal network cascade, and contributes to acomplex series of physiological and pathological events. In many signal transductionpathways, cell skeleton, the first perception of mechanical structure, plays an importantrole. Osteoblast reaction to external mechanical force signal is mainly through F-actin inresponse.The interaction between F-actin and trans-membrane molecules is the main linkof signal transmission. Osteoblast cell skeleton reconstruction depends on the sensit ivityregulation of mechanical key protein function. ADF/Cofilin family proteins are uniquedepolymerization of actin binding protein group, the actin plays an important regulatoryfunction: when the actin filament activates, ADF/Cofilin can cut off the actin filamentfamily protein, which in turn increases muscle actin monomers solution polymerization ofthe end of wire. This suggests that cofilin plays an important role in signal transduction.While previous studies have focused on endothelial cells, nerves, muscle and so on, osteoblast has been rarely studied.This experiment is set based on Flexcell stress loading system, MG-63cells withdifferent loading time cyclical tension. Techniques including immunofluorescence.RT-PCR, Western Blot, gene silencing technology are utilized to explore the MG-63cellsby holding tension stimulation Cofilin expression, as well as in the normal expression,cofilin gene silence, and its influence on the distribution of purpose gene upstream anddownstream, which reveals the role of cofilin in mechanical stimulus cell signaltransduction, providing a new basis for biomechanics of bone transduction mechanisms,and new ideas on accelerating bone defect,as well as providing new targets for screeningand treating of bone metabolic imbalance related diseases.1.An Experiment Study on the Expression Change of Cofilin on theMG-63Cell induced by Mechanical StrainObjective：To investigate the expression change of mechanical strain on cofilin inMG-63osteoblastic cells and to preliminarily explore the mechanism of the cofilin on thecell mechanics signal transduction.Methods：Mechanical strain(12%) was applied to MG-63for0(control group)，1，4，8，12，24hour in vitro．Fixing Cells immediately and immunofluorescence was utilized tofurther process it. Cofilin mRNA expression was examined by RT-PCR. Western Blottingwas used to detect the expression of cofilin.Results：The fluorescence intensity of cofilin was enhanced from0to4hours, thendecreased from8to24hours. However, the expression of cofilin mRNA has no significantchange with mechanical stain (p>0.05). The expression of cofilin in MG-63cells waspromoted by12%mechanical strain elongation. This expression level was graduallyincreased within the first4hours of mechanical strain application (p<0.05). Theexpression level of cofilin was then decreased in8、12and24hours (p<0.05)，but stillhigher than control group.Conclusions：These results indicate that cofilin has significantly expression in MG-63cells on the mechanical strain elongation. Consequently, cofilin may play an importantrole in cell signal transduction.2.Construct Low Cofilin Protein Expression ModelObjective: In order to make a clear definition of the role of cofilin, this study used the shRNA interference carrier to construct cofilin lower expression model to discuss theaffects of cofilin on the osteogenetic differentiation.Methods: Different cofilin shRNA fragments were designed, followed by transfectionof MG-63cells. Fluorescence microscope was used to observe cell transfection, cofilinmRNA expression was examined by RT-PCR. Western Blotting was used to detect theexpression of cofilin.Results: The four segments of interference at the mRNA level significantly reducethe level of cofilin (p<0.05), but no significant difference between each segment wasobserved (p>0.05). Western blot results show that the no.1and no.4pieces significantlylowered the content of cofilin (p <0.001).Conclusion: The no.1and no.4shRNA interference segments can inhibit theexpression of cofilin.3.Effects of Cofilin on the Mechanical Strain Induced OsteogeneticAffect of OsteoblastObjective: By observing the normal expression of cofilin cells and inhibitingexpression of cofilin cells under the mechanical stimulus on the osteogeneticdifferentiation and osteogenesis related genes mRNA level, to confirm cofilin in the roleof mechanical stimulation osteogenesis,to explore cofilin in promoting osteoblastosteogenetic effect of mechanical stress mediated upstream and downstream of themolecular mechanism of signal transduction mechanism provides new evidence forosteoblasts mechanics.Methods: Cells were divided into normal expression cofilin group and cofilin inhibitinggroup.Same mechanical strain was used to stimulate cells. ALP, OCN, COL1, Runx-2mRNA levels were examined by RT-PCR.Results: Cofilin normal expression after mechanical stimulus ALP,OCN,COL1,Runx-2gene mRNA levels have obvious changes.The expression level was higher than controlgroup (p<0.05).In cofilin inhibition group, ALP,OCN,COL1mRNA expressions aresignificantly lower than control group (p<0.05),while Runx-2mRNA expression has nosignificant difference (p>0.05).Conclusion: Cofilin can significantly promote osteogenesis gene activation under themechanical force.When cofilin inhibited, it can not prompt the osteogenetic activity.Cofilin as one of the cytoskeleton regulatory proteins plays an important role incell mechanics signal transduction.