| Background Cerebrovascular disease has become one of the major diseases endangering the health and life of the elderly in our country.Stroke is a major public health problem facing all countries in the world.Stroke has now become the second leading cause of death in humans and the third most common cause of disability,which posing a tremendous financial burden to society and home.In our country,stroke has surpassed cardiovascular disease as a disease with the highest morbidity,lethality and morbidity.Ischemic stroke is one of the major types of stroke.In our country,more than 70% of ischemic stroke patients are newly stroked each year.Interventional therapy is the moment to solve an important means of cerebrovascular disease.Carotid artery as one of the main surgical interventional pathway and intervention of the common carotid artery can cause injury of the common carotid artery.It is well-known that arterial endothelial injury can cause serious consequences such as platelet activation,inflammatory response,local or systemic hypercoagulability and even vascular restenosis.Repair of carotid artery endothelial injury caused by interventional therapy has become an urgent problem to be solved in clinical and research hot spots.Exosomes are membrane-derived vesicles that are 40-100 nm in diameter and are distributed in a variety of bodily fluids,and various tissue cells release exosomes under resting or agonistic conditions.Exosomes are important carriers of cell autocrine and paracrine and have important potential value in clinical treatment.Exosomes contain proteins,m RNA,micro RNAs(mi RNAs),among them mi RNAs have a variety of roles and involved in all aspects of life activities.And it has been the current research hot spots.Endothelial progenitor cells are precursor cells of vascular endothelial cells,also called hemangioblasts.Studies have reported that exosome derived from endothelial progenitor cells can repair vascular endothelial injury.While the exosomes derived from endothelial progenitor cells on the common carotid artery endothelial injury repair and repair mechanism remains to be further studied.Objective(1)In this study,C57 BL / 6 mice were used as research objects,and C57 BL / 6 mice were used to construct common carotid artery endothelial injury in animal models to clarify endothelial progenitor cells exocrine can repair carotid artery endothelial injury.(2)To investigate the repair mechanism of endothelial injury in the common carotid artery by the components(mainly micro RNA)contained in the exosome secretion of endothelial progenitor cells.Method(1)Cell immunofluorescence experiment Induction of differentiated endothelial progenitor cells and primary culture of common carotid artery endothelial cells were incubated with the corresponding primary antibody: CD133,Fl K-1 and Factor Ⅷ,VEGF,and then incubated with secondary antibodies,photographed under a fluorescence microscope.(2)Projection electron camera The freshly extracted exosomes of endothelial progenitor cells were placed under transmission electron microscope to observe the diameter and morphology of the exosomes.(3)Western blotting experiments Exosomes from freshly extracted EPCs were lysed,electrophoresed,transferred to membrane and exposed to detect CD9,CD63 and CD81 protein bands.(4)Endothelial progenitor cell exosome mi RNAs high-throughput sequencing The newly extracted exosomes of endothelium progenitor cells were entrusted by the company to build the database and go through the on-line sequencing to obtain the original data.Later,the data were filtered and differentially clustered,and the total expression,target gene prediction and KEGG analysis were performed.(5)Cell scratch test The carotid artery endothelial cells were treated with exosomes of endothelial progenitor cells and photographed after 0 hour and 12 hours to analyze the changes of cell proliferation and migration ability.(6)Vascular Tension Experiment The mice in the carotid artery endothelial injury model group and the exosome group in which endothelial progenitor cells were excreted were sacrificed and the common carotid arteries were removed for vascular tone test to detect the changes of endothelium-dependent relaxation and endothelium-independent relaxation.(7)Immunohistochemistry experiment The mice in model group of carotid artery endothelial injury and the exosomes in endothelial progenitor cells were sacrificed and the common carotid arteries were removed for immunohistochemistry to detect the change of VEGF expression in the endothelium.Results(1)The result of immunofluorescence showed that the protein markers of endothelial progenitor cells induced by differentiation: strongly positive for CD133 and Fl K-1;The exosomes of endothelial progenitor cells showed a typical "cup-torch" shape under the electron microscope with a diameter of 40-100 nm.Western blot results showed clear bands of CD9,CD63 and CD81;Immunofluorescence results showed that primary culture of common carotid artery endothelial cell markers: Factor Ⅷ,VEGF was strongly positive.(2)Endothelial progenitor cell exosome mi RNAs high-throughput sequencing showed that endothelial progenitor cells exosomes rich in micro RNA: mmu-mi R-146a-5p,and through target gene prediction and KEGG analysis found that mmu-mi R-146a-5p can act on VEGF signaling pathway.(3)Scratch assay showed that exosome secretion of endothelial progenitor cells could significantly enhance the proliferation and migration of carotid artery endothelial cells.The experimental results of carotid artery vascular tension showed that exosomes of endothelial progenitor cells could significantly improve the endothelium-dependent vasodilation of injured endothelium.The results of immunohistochemistry showed that exosomes excreted by endothelial progenitor cells could increase the expression of VEGF in the common carotid artery endothelium in the model group.Conclusion(1)Endothelial progenitor cell exosomes can promote the proliferation of endothelial cells of the common carotid artery and up-regulate the function of endothelial cells to repair the common carotid artery endothelial injury.(2)The mechanism of endothelial progenitor cells excretion on the common carotid artery endothelium may be related to the micro RNA: mmu-mi R-146a-5p,which is activated by VEGF in the common carotid artery endothelial cells and promotes the common carotid artery Endothelial injury repair. |
PDF Full Text Request