Fluorescence Resonance Energy Transfer (fret) Method Of Leptin And External Stimuli For Cartilage Cytoskeletal Regulatory Proteins Rhoa

Background:Osteoarthritis is a kind of degenerative disease,which represents one of the most frequent diseases encountered in the clinics.However,the pathophysiology of OA is still unclear.It is believed that obesity represents one of the significant risk factors for OA.Many studies have demonstrated a clear link between obesity and OA.Leptin is a small polypeptide excreted by white adipose tissue, whose circulating concentrations are proportional to the degree of adiposity.In 2003, Dumond et al.found that leptin was strongly overexpressed in human OA cartilage and synovial fluid,which indicates leptin may play a key role in the pathophysiology of OA.RhoA is a significant molecular switch,which regulates a wide spectrum of cellular functions such as remodeling of the actin cytoskeleton.Many studies showed that OA chondrocytes have an elevated level of cytoskeletal component.Our research hypothesizes that leptin and external force may affect the activity of RhoA in normal chondrocytes,consequently induce some changes of the cytoskeleton.We believe that fluorescent resonance energy transfer(FRET) could be a proper method to demonstrate the changes of RhoA in a single living chondrocyte.Methods:pRaichu-1237x vector,encoding YFP-RhoA-PKN-CFP fusion protein, was transfected into normal chondrocytes via Human Chondrocyte Nucleofector^TM Kit(Amaxa Corporation,Germany).Chondrocytes were incubated with 200ng/ml leptin for 30 minutes.The FRET signal of RhoA was recorded during the incubation. Besides,we also used AFM as an apparatus to apply external force to the chondrocytes,with the purpose of observing the change of RhoA FRET signal.Results:During the incubation,we observed a pseudopodium contraction and cytoplasm streaming.The FRET signal gradually vanished.The whole chondrocyte collapsed in the end.Many small bubbles could be found around the plasma membrane.After applying external force to the chondrocytes by AFM,we observed an obvious enhancement of the RhoA FRET signal.Cell accepted a tapping mode of external force also appeared an obvious pseudopodium contraction,whereas cell accepted a contact mode of external force did not show any change of the outline.Conclusions:1.Incubating chondrocytes with high-dose leptin(200ng/ml) could not activate RhoA,but lead to FRET signal vanishing.Cell collapse and small bubbles around the membrane may indicate cell apoptosis.2.AFM has been proved to be a successful method to apply external force to the living cells.Tapping mode and contact mode could simulate pressure and shearing force separately.It seemed that chondrocytes were more sensitive to the tapping mode,which induced stronger RhoA FRET signal and more visible change of the cell outline.All the evidences show that external force could obviously activate RhoA...