Effect Of Exosome Derived From HRMVPC-Immortalized On Tubular Formation Of High Glucose HRVECS

BackgroundDiabetic retinopathy(DR)is an important disease that causes blindness in China,but the recent research on its pathogenesis is still in the stage of hypothesis and verification.Neovascularization is considered to be the most serious stage in the development of diabetic retinopathy.For a long time in the past,the study of microvessels is mainly focused on endothelial cells,while the study of microvascular pericytes lags behind.However,pericytes also play a key regulatory role in a variety of physiological and pathological processes of microvessels.When a variety of factors(such as high glucose environment)lead to pericyte apoptosis or exfoliation from microvascular position,it will increase microvascular permeability,cause hemorrhage and vascular swelling,and further induce edema,DR and other diseases.In addition,pericytes also play an important role in supporting microvascular structural stability,promoting neovascularization and vascular remodeling.The distribution of pericytes is different in organs and tissues,and the number of microvascular cells in different organs and tissues is different.It can be seen that pericytes are very important for the regulation of retinal microenvironment.Recently,exosomes have been proved to have intracellular/intercellular regulation,and some studies have been carried out on oxidative stress,chronic inflammation and neovascularization,which have great biological activities in the mammals.However,it has not been deeply studied in retinal diseases and its related pathway mechanism.mi RNA and other active substances secreted by retinal cells such as Muller cells and microglia may play a role in regulating retinal neovascularization.The study of this mechanism and pathway can provide a new direction and more early intervention and early warning mechanism for the treatment of DR.At the same time,it can also provide a new micro-perspective for the pathogenesis of DR.ObjectiveTo abstract and classify exosome derived from pericytes,and study its effect on retinal vascular cells,and establish a tube model of retinal endothelial cells,so as to provide a basis for early diagnosis and early intervention of DR.Methods1.The exosome was extracted from the supernatant of pericyte culture by differential centrifugation,and the exosome was identified by electron microscope,exosome staining,particle size analysis and Western Blot specific CD63.2.Detection of mi RNA expression in exosome bodies derived from pericytes by second-generation sequencing.3.Through a model of retinal vascular endothelial cell formation was established to simulate the process of retinal neovascularization,and the effects of high glucose,low glucose and exosome on vascular tube formation were examined by migration,proliferation and apoptosis test.4.q PCR test were used to verify the expression of corresponding factors in the experimental group.Results1.The exosome was extracted by pericytes,and the electron microscope results showed that the exosome structure was obvious.The particle size analysis showed that most of the particles in the sample were in the range of 60-100nm,which conformed to the exosome characteristics.Flow analysis showed that the expression rate of CD63in the sample was high.2.In the study of the effect of high glucose environment on retinal endothelial cells,the DMEM culture medium of low glucose group(15mmol/L)and high glucose group(50mmol/L)was used respectively.The angiogenesis experiment showed that the number of vascular nodes and tubules in the high glucose group was more than two times higher than that in the low glucose group;q PCR assay showed that the expression of VEGF-A m RNA in the high glucose group was more than three times higher than that in the low glucose group,and the expression of Hif-1αm RNA in the high glucose group was more than two times higher than that in the low glucose group.The migration experiment showed that the migration rate in the high glucose group was more than twice as highas that in the low glucose group.The proliferation experiment showed that the proliferation of retinal vascular endothelial cells in the high glucose group was higher than that in the low glucose group at 24 hours,48 hours and 72 hours,and the flow cytometry showed that the apoptosis in the high glucose group decreased by about 20%.3.In the study of the effect of pericyte exosome on retinal endothelial cells,high glucose group,high glucose+PBS group and high glucose+exosome group(1μg/10~6cells)were used respectively.The exosome staining experiment showed that only the exosome group showed fluorescence,and the angiogenesis experiment showed that the number of vascular nodes and tubules formed by the exosome were significantly decreased(P<0.01).q PCR assay showed that the expression of VEGF-A m RNA and Hif-1αm RNA decreased in exosome group(P<0.05).The migration experiment showed that the migration rate in the exosome group decreased significantly(P<0.001).Proliferation test showed that the proliferation of vascular cells in exosome group was lower than that in high glucose group and high glucose PBS group at 24 h,48h and72h(P<0.01).Flow cytometry showed that vascular endothelial cell apoptosis increased by about 10%in exosome group.4.The results of second generation sequencing showed that there were 36 kinds of up-regulated mi RNA and 140 kinds of down-regulated mi RNA in exosome samples.The enrichment of Gene Ontology(GO)showed that there were enrichment of nuclear and cell membrane genes in cell components,RNA regulatory genes and DNA transcription genes in biological function types,and ATP,RNA binding genes with metal ions in molecular function types.The bubble diagram shows the enrichment of genes related to VEGF pathway,c AMP pathway,c GMP-PKG and oxidative stress pathway.ConclusionsThe exosomes secreted by pericytes can effectively inhibit the excessive formation of retinal blood vessels in the environment of high glucose,and the possible mechanism is the regulation of mi RNA in the exosomes in the VEGF pathway.Therefore,exosomes have the potential to interfere or predict the occurrence and development of early DR.