Construction Of Directional Stress-fiber Cell Model And Study The Response Of Cell To Stress,Rearrangement Of Stress Fiber And Rho

The status of the substrate have a major influence on the growth of cell and the morphology of cell decides its life and death. So the cellular mechanotransduction has been a hot research issue. The research of mechanotransdution is divided into two aspects:mechano signal and chemical sinal.But there is no one authority theory to completely explain how the mechanical signal transfer .This paper have preliminarily study the influence of static strain to cytoskeleton and the distribution of Rho protein in the cell by built a directional live cell model of stress filaments in the aspect of response of cytoskeleton to mechanical signal.The results of study provided a new way to research the relation between cell and direction of force and have some significance on the to further explain the response of cell to stress,rearrangement of stress fiber and signal transduction of Rho protein.The main results of study is following:①Design of mechanical loading device:We have designed an uniaxal loading device according to the substrate straining principle.The device have 3 components: control unit, mechanic unit and culturing chamber.It can provide the uniaxial cyclical straining style.The integrate evaluation of system suggests: system operates stably, the temperature can be controlled, the frequency and deformation(frequency:0.01-1.5HZ, strain 0-50%) can be regulated easily, the duration can be controlled and aseptic operation is simple. It can be used to different frequency, strain and duration stretch experiment.②Construction of directional stress fiber cell modelAccording to the study of cell cyclical loading by previous paper, We have loaded the ECV-304 by cyclical straining and studied the change of the cell morphology and stress filament after a period of time. We confirmed the direction of force's influence on the direction of short and long diameter respectively and stress filament. It suggests that: The cell morphology and the parallel-arranged manner in the cell can maintain stability in a period of time after the cyclical straining stopped. The method of build a directional live cell model which is stable in the period time by cyclical straining is reliable. This model can be used to further study the influence of force to cell orientation.③The static straining of directional stress filament cell model We imposed 10% of the static straining on cell model in parallel with the direction of stress fibers. The results suggest that the response of the cell to the loading direction is contraction in the 30 minutes and the response of the vertical direction of loading had no significant change and in the same time the bunch structure of stress filament is disappearing gradually, the filament got blurred and depolymerized.Accrding to the relative report and combined with our own experiments, this phenomenon can be explained as:1. The excessive straining of substrate leaded to the adhesion structure torn from substrate so the cell retracted.2Because excessive stress through the skeleton structure transmitted into the cells triggered the double-related confrontation adjustment mechanism of Rho protein. By the feedback regulation of stress the stress filament depolymerized and cell retracted.④Study on the expression of Rho protein in cellWe study the expression of Rho protein by sampled cells from static group and cyclical straining group which are treated by immunofluorescence technique. We found that Rho distributed in the cytoplasm and nuclear and the distribution had not obvious change which can be discernible by the naked eye after cyclical straining. The immunOfluorescence intensity of Rho protein is relative low in the static cells.We have statisticsed the light intensity value by the image processing software—Quantity One and found that fluorescence intensity of Rho protein in the cell increased after cyclical straining.They had significant difference by T-test(P<0.01).It suggested that amount of Rho protein increased after cyclical straining. Because the non-specific staining structures appeared in the nuclear area,it have major influence on the measurement of light intensity of short diameter and long diameter direction in the cells so it is hard to obtain the difference of directions.We found that Rho protein was no significant difference in the long and short diameter directions after static straining or cyclical straining.