Effects Of PRP On Migration And Invasion Of The RA-FLS

Background and Objective:Platelet-rich plasma (PRP) is the blood plasma that has been enriched with platelets. PRP, activated by 10% calcium chloride and bovine thrombin, releases many different growth factors and other cytokinesis, including platelet-derived growth factor (PDGF), transforming growth factor-(TGF-?), vascular endothelial growth factor (VEGF) and insulin-like growth factor-1 (IGF-1), which play important roles in cell proliferation, differentiation, bone and soft healing and collagen synthesis.Rheumatoid arthritis (RA) is a chronic autoimmune disease, characterized by inflammatory cells infiltration and synovial hyperplasia, eventually leading to joint destruction and disability. Fibroblast-like synoviocyte (FLS) plays an important role in initiating and driving of RA. RA-FLS can secrete metalloproteinases (MMPs), adhere to the extracellular matrix (ECM), migrate and invade into the cartilage, and finally exacerbate joint damage. Several studies have shown that the account of platelets correlates with the rheumatoid activity. The aim of this study was to investigate the effects of PRP on migration and invasion of the RA-FLS.Methods:1. PRP was activated by thrombin and calcium chloride, and centrifuged and the supernatant was used for experiments.2. Wound-healing and transwell migration and invasion assays were performed to study the effects of PRP on the migration and invasiveness of RA-FLS.3. Cell-matrix adhesion assay was used to test the ability of RA-FLS adhesive onto the ECM.4. Immunofluorescence assay was applied to evaluate the effects of PRP on the actin cytoskeleton rearrangement and the formation of filopodia and lamellipodia in RA-FLS.5. Real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR) was used to examine the levels of MMPs mRNA of RA-FLS.Results:1. Wound-healing assay showed that PRP promoted the migration of the RA-FLS. The transwell cell migration assay demonstrated that the number of RA-FLS across the polycarbonate membrane in the 2% and 5% PRP group was 2.02±0.78,2.59±0.90 respectively relative to the NC group(P<0.05).2. Transwell invasion assay showed that the number of RA-FLS across the Matrigel barrier in the 2% and 5% PRP group was 1.50±0.22,2.68±0.35 respectively relative to the NC group(P<0.05).3. Cell adhension to collagen type I matrix assay showed that the number of RA-FLS treated with 2% and 5% PRP adhesive onto the collagen type I was 1.50±0.27, 1.87±0.62 respectively relative to the NC group (P<0.05).4. The results from the immunofluorescence assay showed that PRP induced a significant decrease the number of centrally located stress fibers and caused an increase of formation of filopodia and lamellipodia in detectable leading edge in the RA-FLS.5. RT-qPCR assay showed that PRP can upregulate the levels of mRNA of MMP-1, MMP-3 and MMP-10. Conclusion:1. PRP promotes the cell-matrix anhesion, migration and invasion of RA-FLS.2. Promotion of RA-FLS cell migration, invasion, and adhesion on matrix by PRP might be modulated through its up-regulating MMP-1, MMP-3 and MMP-10 mRNA expression and the actin cytoskeletal reorganization.