Protease-Activated Receptors 1&4-Stimulated Platelet Releasates Promote Injured Tendon Repair:An Experimental Study

Part 1. Preparation and characterization of protease-activated receptors1&4-stimulated platelet releasateObjective: To explore the in vitro activation of PAR1 & 4 optimization plan, prepare two stable release liquid receptor activation methods; identification PAR1 & 4-PR in each different cell / growth factor content.Method: First optimization PAR1 & 4-PR programs to find the best activation activating protease activated receptor concentration, obtained PAR1 & 4-PR by VEGF and Endostatin detect whether successful activation and activation efficiency. Then fix related content via cell factor / growth factor antibody microarray PAR1 activation and release liquid tendon 4, and then select which differences are significant important factor ELISA test verification and validation.Results: The optimum concentration of activator to activate PAR1 was 20μM, and activate PAR1 activators optimal concentration of 10μM. Endostatin and VEGF by ELISA kits detect PAR1-PR indeed mainly rich VEGF and PAR4-PR major rich Endostatin. Verify results of cell / growth factor antibody microarray results and ELISA experiments are shown PAR1-PR in VEGF, EGF, MCP-3, IL-8, GRO-α, the higher the content of MMP-9,and contains more PAR4-PR the Endostatin, FGF, TGF-β.Conclusion: We successfully prepared adult PAR1 & 4-PR and optimize their preparation programs, and detected a major release of two kinds of cells were associated with tendon / growth factor differences. PAR1-PR contains more promoting angiogenesis factors, pro-inflammatory cytokines and promoting catabolism factor(VEGF, EGF, MCP-3,IL-8, GRO-α, MMP-9), and in PAR4-PR contains more inhibiting angiogenesis and promoting cell proliferation factor(Endostatin, FGF, TGF-β)Part 2. In vitro studies on the different effects of PAR1&4-PR on adult tendon stem cells(TSC) and endothelial progenitor cells(EPC)Objective: To explore the PAR1 & 4-PR on two key cells(adult TSC and EPC)tendon repair process different effects from in vitro study was to explore the role of two kinds of release in tendon repair process may play.Methods: we extracted from adult tendon tissue and peripheral blood identified adult TSC and EPC, and then examine the effects of PAR1 & 4-PR on both cell proliferation and migration by CCK-8 experimental method, and then by q RT-PCR, Sircol, ELISA and Western Blot and other methods to study the release of protease activated receptor expression was on TSC differentiation metabolism and inflammation and other related genes and proteins. Effect of vascular again into two kinds of EPC release liquid into the blood vessel through the EPC capability in vitro.Results: First, we successfully extracted and identified from the TSC adult tendon tissue, in vitro experiments showed that: proliferation assay showed that PAR4-PR than PAR1-PR is stronger proliferation, migration experiments show that both of TSC no significant role in promoting the migration, while the protein and genomic results indicate that PAR4-PR can induce more TSC to tendon cells and activated cells to synthesize a higher metabolism, and can produce more collagen synthesis; and PAR1-PR has some proliferation capacity but lower than the former, can induce cell is at a higher level of catabolism and inflammation levels. We also successfully extracted from human peripheral blood and identification of the EPC, in vitro experiments showed that PAR1-PR ability to promote EPC proliferation stronger than PAR4-PR. Migration assay then displays PAR1-PR more than promote migration of PAR4-PR. In vitro angiogenesis experiments also showed, PAR1-PR form stronger than the pro-EPC cultured vascular PAR4-PR’s.Conclusion: TSCPAR4-PR has more proliferation, differentiation-promoting,anabolic effect, and PAR1-PR to promote higher levels of inflammation and catabolism.For EPC, PAR1-PR has better proliferation, migration, and promoting pro-angiogenesis effect.Part 3. In Vivo study on the application of PAR1&4-PR to promote injured tendon repairObjective: To investigate the in vivo effects of PAR1 & 4-PR to promote tendon repair.Methods: In this part, we first prepared PAR1 & 4-PR follow the previous steps, and then in New Zealand white rabbits were manufactured right knee tendon injury model, then randomly divided into three groups were given PAR1-PR, PAR4-PR and saline. Four weeks after drawn histological sections were stained, q RT-PCR, Western Blot and biomechanical assay PAR1 & in vivo therapeutic effect of 4-PR.Results: Tissue HE and immunohistochemical staining showed that the tendon tissue repair after PAR4-PR treated more neatly arranged and no obvious positive staining of CD31 and CD68, and tendon tissue repair after PAR1-PR treated the disorder, and the organization It shows multiple CD31 and CD68 positive blood vessel-like tissue. Gene and proteomic experimental results show that PAR4-PR can promote more Col-1 Col-3expression. Biomechanical Experimental results show that the mechanical properties of tendon tissue repair after PAR4-PR treated higher than PAR1-PR-treated group and the control group.Conclusion: PAR1 receptor activation release fluid can effectively promote the repair and remodeling of tendon tissue structure and increase the mechanical properties of tendon tissue; and PAR1-PR can induce angiogenesis in more tendon tissue, reduces tendon tissue mechanical properties.