The Experimental Studies Of Endothelial Progenitor Cells Drive Dexosome Sonvascular Repair After Traumatic Brain Injury In Mice

Objective: Traumaticbraininjury(TBI)is a serious hazard to human health,with high morbidity and mortality.The prognosis of patients after traumatic brain injury is determined by the repair of nerve and blood vessels.Our previous study confirmed that endothelial progenitor cells(endothelial colony forming cells,EPCs)mobilization increased at first and then decreased in the peripheral bloodafter traumatic brain injury,there is also evidence of endothelial progenitor cells capable of homing to the local traumatic lesions around the focus and promote angiogenesis,but the endothelial progenitor cells whether directly differentiate into vascular endothelial cells of the mechanism involved in angiogenesis is still unclear.Previous reports have proved that endothelial progenitor cells promote the injury of the vascular endothelial cell proliferation and differentiation of the stump formation of angiogenesisthrough paracrine actions,but not directly involved in the repair of differentiation into vascular endothelial cells.Endothelial progenitor cells have been shown to secrete exosomes,a variety of biological substances and endothelial progenitor cell exosomes contain the internal,but the exosomes derived endothelial progenitor cells in promoting the nerve and vascular repair after traumatic brain injury is not clear.This experiment through the observation of endothelial progenitor cells exosomes in vivo or intro of damage endothelial cells model,to provide a theoretical reference for the application of endothelial progenitor cell exosomes treatment of traumatic brain injury.Methods:(1)To culture and purification of endothelial progenitor cells and identify its function from umbilical cord blood mononuclear cells in vitro by gradient density centrifugation method(MNCs),;(2)To obtained endothelial progenitor cell exosomes in vitro secreted into the culture medium by ultracentrifugation method,We application method of transmission electron microscope and Western blot observed morphological characteristics of endothelial progenitor cells in exosomes and surface specific protein molecules.(3)The endothelial cell injury model was established in vitro by hypoxia incubator.The expression of blot and the expression of the protein were detected by Western.(4)The application of brain cortex injury instrument(CCI)preparation of craniocerebral injury in mice,transplantation of endothelial progenitor cell exosomesby mice tail vein,using immunofluorescencemethods and Western blotdetection of wound around the focus of microvascular density changes of tight junction proteins after endothelial progenitor cell exosomes transplantation into TBI model.We also measure theleakage of Evans blue,through Blood brain barrier.The PCR was used to detect the expression of tight junction protein,The application of Western blot technique to detect angiogenic protein PTEN expression.Results: We obtain the umbilical cord blood mononuclear cells in vitro by gradient density centrifugation method successfully,and were cultured in vitro;By ultracentrifugation method to obtain endothelial progenitor cellsexosomes,Through transmission electron microscope and Western blot method we identification of endothelial progenitor cells can secrete exosomes;We also labeled exosomes with fluorescent dye PKH26,confirmed that endothelial cells could engulf progenitor cell exosomes;In vitro endothelial progenitor cells exosomesincreased injured endothelial cell tight junction protein CLN5,ZO-1,OCLN expression,decreased PTEN expression,increasedp-Akt level.In vivo experiments suggest that umbilical cord blood endothelial progenitor cells exosomes can reduce of blood brain barrier permeability by Evans blue permeationexperiment after traumatic brain injury;The PCR technology confirmed that tight junction protein CLN5,ZO-1,OCLN expression increased around traumatic lesions of brain,the VEGF expression increased,and the MMP9 expression levels increased;Western blot shows that exosomes can reduce the expression of PTEN level at the third days and the seventh days after TBI.Conclusion:Endothelial progenitor cells can secrete a large number of exosomes.In vitro andin vivo,endothelial progenitor cellsexosomes can promote endothelial cell proliferation and repair after injury.The exosomes also increased the expression levelof vascular repair proteins after traumatic brain injury,The direct transplantation of endothelial cell exosomes is more safe and effective thantransplantation stem cell and the clinical application prospect is Broad,but still need basic and clinical research of exosomeneed further study...