The Effects Of Mechanical Stimulation On Differentiation Of Osteoblasts Promoted By Bone Mesenchymal Stem Cells And Expression Of Pannexin1and The ATP Release

Objective Investigate the effect of stress stimulus on alkaline phosphatase activity.Observe the expression of collagen protein I with stress stimulus. Observe theexpression and distribution of pannexin1protein of intracellular with stress stimulus.Observe of stress stimulate cells to release ATP changes.Methods1.Healthy3-weeks-old SD rats of both sexes weighing from100to120g. Theprimary marrow stromal cells of the femur and tibia of the rats were procured andcultured.2.Violet spectrophotometer method:The mesenchymal stem cells were randomlydivided into two groups: applied stress stimulation group and control group. The appliedstress stimulation group was irritated for15minutes,4000μstrain.After the cells digestedthe number were counted at once. Smash the cells by sonicator and the alkalinephosphatase was analyzed by the violet spectrophotometer.3.Immunohistochemistry: The mesenchymal stem cells were randomly divided intotwo groups: applied stress stimulation group and control group.Observe the expressionof collagen protein I.4.Observe the expression and distribution of pannexin1protein using the confocallaser scanning microscope.5.Fluorescein enzymatic:The rats were divided into three groups: blank group,applied stress stimulation group and control group. Observe the changes of ATP releaseusing the biological fluorescent reagents.6.Data analysis was performed by SPSS16.0.The group comparison adoptedone-way ANOVE. P<0.05was considered statistical significance. P<0.01wasconsidered significantly statistical significance. Results1.MSCs adhered to the petri dish after24h grew well.After48h cell coverage ratewas around70%to80%. Cultured cells could stably grew and passage.2.Violet spectrophotometer method The alkaline phosphatase activity wassignificantly increased in stress stimulation group.3.Immunohistochemical The expression of collagen protein I was significantlyincreased in stress stimulation group.4.Fluorescence microscope It can be observed that the expression anddistribution of pannexin1protein was changed.5.The release of ATP was significantly increased in stress stimulation group.Conclusion1.The activity of alkaline phosphatase and the expression of collagen protein I wasincreased in stress stimulation group.And that promoted the differentiation ofosteoblasts.2. The expression and distribution of pannexin1protein was obvious increased instress stimulation group.3. The release of ATP was significantly increased in stress stimulation group.