Study On The Treatment And Mechanism Of Platelet-derived Exosomes In Diabetic Wounds

PART Ⅰ COMPARISON AND INVESTIGATION OF EXOSOMES DERIVED FROM PLATELET-RICH PLASMA ACTIVATED BY DIFFERENT AGONISTSObjective: To select the best PRP activator to obtain high quality exosomes for the next step of research.Methods:(1)After PRP was activated by different activators(calcium gluconate,thrombin or both),PRG was collected and HE staining was performed to observe the fibrin distribution.Exosomes were obtained from supernatant by gradient hypercentrifugation.(2)The differences in the quantity,size and growth factor content of exosomes between different groups were compared and analyzed by TEM,Nanoflow and WB.Results:(1)The rate of PRP activation by thrombin was much higher than that of calcium gluconate,and the effect of combination of thrombin and calcium gluconate was better than that of single activator.(2)By TEM,Nanoflow,WB detection,the sediments in each group were identified as exosomes.(3)TEM,nanometer flow,WB results showed that the mixture of calcium gluconate and thrombin could activate PRP to release exosomes with the highest concentration,and the exosomes contained the highest concentration of growth factors(VEGF,PDGFBB,BFGF,TGF-β).Conclusion: The mixture of calcium gluconate and thrombin is superior to a single activator and can activate PRP to release high concentration and high quality exosomes.PART Ⅱ STUDY ON THE TREATMENT AND MECHANISM OF PLATELET-DERIVED EXOSOMES ON WOUND SURFACE IN TYPE 2 DIABETIC RATSObjective: Our previous studies have proved that PRP contains a large number of growth factors,which can promote wound healing in diabetic foot patients,but the underlying mechanism is still unclear.In recent years,studies have shown that platelets will release a large number of exosomes after activation.Exosomes play an important role in cell-to-cell communication and have good prospects in cell-free therapy.This study aims to study the effect of PRP-derived exosomes on diabetic skin wound healing and its underlying mechanism in a diabetic rat modelMethods: 1.Separation and identification of exosomes: Use gradient ultracentrifugation to separate exosomes from PRP.TEM,Nanoflow and western blot were used to identify exosomes.2.Animal experiment: use streptozotocin-induced diabetic rats to establish a full-thickness skin trauma model.The rats were randomly divided into a control group and a PRP-Exos group,with 10 rats in each group.PRP-Exos was injected dispersedly on the edge of the wound,and the control group was injected with the same amount of PBS.Observe and evaluate the effect of PRP-Exos on inflammation and wound healing.The tissues were analyzed histologically by HE staining,immunofluorescence and immunohistochemistry.3.Cytological experiments: HUVEC,HDF were used for cytological analysis.Through immunofluorescence,CCK8,transwell,tubule formation,Western blot analysis of cellular exosomes uptake,cell migration,cell proliferation,and the use of signal pathway inhibitors to further verify the effect of exosomes on cell signal pathways.Results: The results of animal experiments showed that compared with the control group,PRP-Exos can significantly promote skin wound healing(p <0.05).The results of HE staining,Masson staining,immunohistochemistry,and immunofluorescence showed that compared with the control group,PRP-Exos promoted wound healing,increased collagen deposition,improved wound matrix composition,and created more skin appendages.Contribute to the formation of scarless wounds.Cytological experiments showed that PRP-Exos can stimulate the proliferation and migration of HDF and HUVEC in a dose-dependent manner.The optimal dose is 50ug/ml.In addition,compared with the blank control group and the inhibitor group,the PRP-Exos treatment group significantly activated the Akt signaling pathway,promoted the production of HDF type I and III collagen,and inhibited the expression ofα-SMA protein(p <0.05).Conclusion: Our experimental results show that platelet-derived exosomes may promote the healing of diabetic ulcers by activating the Akt signaling pathway,and at the same time improve wound remodeling,providing a new method for the future treatment of diabetic wounds.