ABSTRACT
Mechanical pressure plays an important role in many physiological and pathological processes. Mimicking the mechanical pressure present in vitro is necessary for related research, but usually requires expensive and complicated equipment. In this study we created a simple pressure culture system based on the transwell culture system. By cutting off the top rim of the transwell insert, the cells were compressed between the insert membrane and the well floor. The new pressure culture system was proven effective in that it induced cell morphological change, integrin β1 upregulation, actin polymerization and growth change in rat retinal ganglion cells, human nasopharyngeal carcinoma cells and mice embryonic fibroblasts. Though the pressure value is immeasurable and inhomogeneous, the easily available culture system still provides a choice for the laboratories that do not have access to the better, but much more expensive pressure culture equipment.
Subject(s)
Animals , Humans , Rats , /genetics , Cell Proliferation , Cell Culture Techniques/methods , Analysis of Variance , Actin Cytoskeleton/physiology , Cell Line/physiology , Fibroblasts/physiology , Fluorescent Antibody Technique/methods , Hydrostatic Pressure , Methylamines , Nasopharyngeal Neoplasms/pathology , Primary Cell Culture , Real-Time Polymerase Chain Reaction , Retinal Ganglion Cells/cytology , Retinal Ganglion Cells/physiology , Reverse Transcriptase Polymerase Chain Reaction/methods , Stress, MechanicalABSTRACT
Objective: To investigate the expression and distribution of G protein and protein kinase C (PKC) under the mechanical pressure in rabbit mandibular condylar chondrocytes (MCCs) and to study the role of G protein in PKC signalling pathway. Methods:MCCs from two-week-old New Zealand rabbits were cultured. After treatment under continuous pressure of 90 kPa for 60 min or 360 min by hydraulic pressure controlled cellular strain unit, the expression of G?q/11 protein was examined by Western Blot. The expression and distribution of PKC was observed by immunocytochemical staining. Results:Gaq/11 protein in MCCs treated by 90 kPa for 60 min and 360 min was increased by 163.7% and 65.8% respectively(P0.05). PKC in control cells distributed uniformly in the cytoplasm. After been pressed under 90 kPa for 60 min,PKC translocated to the membrane and, partly,into nuclei. When the pressure prolonged to 360 min, PKC distributed uniformly again in cytoplasm. By treatment of G protein inhibitor, the translocation of PKC under 90 kPa of 60 min was not observed. Conclusion:Feasible pressure may promote G protein expression and activate PKC. The activation of PKC signalling pathway is mediated by G protein.