Isolation And Identification Of Exosomes Derived From Adipose Mesenchymal Stem Cells And Its Effect On Carotid Artery Balloon Injury In Rats

Objective:1.ADMSCs-Exo（adipose-derived mesenchymal stem cells derived exosomes）were extracted and thebiological characteristics were identified;2.To observe the effects of ADMSCs-Exoon carotid balloon injury in rats.Methods:1.ADMSCs（adipose derived mesenchymal stem cells）were isolated,cultured,subcultured and identified:ADMSCs were cultured by enzyme digestion method,and the surface specific antigens of the third generation ADMSCs were detected by flow cytometry.Meanwhile,they were induced to differentiate into adipogenic and osteogenic cells.After 3 weeks,the cell differentiation was observed and identified by oil red O and alizarin red staining,respectively.2.ADMSCs-Exo extraction and identification:ADMSCs was cultured with fetal bovine serum of exocrine body for 48 hours,and exosomes were extracted by Exo-spin ^TMmethod,and the exosomes were detected by TEM（transmission electron microscopy）,NTA（nanoparticle tracking analysis）,WB（western blot）.3.To observe the effect of ADMSCs-Exo on carotid balloon injury in rats3.1Establishment and grouping of carotid balloon injury model in rats:Forty-five male SD rats weighing about 300～350g were randomly divided into three groups:the control group,the injury group,and the exosomes group.The control group was cut and sutured by the neck skin,andin the exosomes group and the injury group,the carotid artery balloon injury model was constructed;meanwhile,the veins were treated with exosomes and the same amount of normal saline.The rats were killed on the 14th and 28th day after operation respectively,and the left common carotid artery was taken.3.2The morphological changes of the common carotid artery were observed by hematoxylin-eosin staining.IA（intima area）and MA（mediaarea）were measured by image imaging method,I/M ratio was used to evaluate the level of carotid intimal hyperplasia.3.3Evans blue staining was used to observe the condition of endothelialization and the ratio of endodermis area to total vascular area was used to evaluate the level of vascular injury and repair.Results:1.ADMSCs cells showed adherent growth,mostly long spindle-shaped,partly polygonal,and aggregated growth.The flow cytometry results of the thirdgeneration ADMSCs showed that the positive rates of CD29,CD90,CD34,CD45,and CD31 were respectively 87.6%,97.9%,37.9%,6.2%,0.9%;After 3weeks of induction of lipid formation,fat droplets were observed under the microscope,which were round in shape,partially fused into a mass,full in shape,and orange in oil red O staining;after 3 weeks of osteogenesis induction,cells were seen in disorder,which was unclear,with white high-density substances and light orange in alizarin red staining;it was confirmed that ADMSCs in isolation and culture was in accordance with MSCs（mesenchymal stem cells）.2.Under TEM,ADMSCs-Exo showed a typical circular membrane structure,containing low density materials,The results of NTA showed that most of the particles were in the range of exosomes diameter（30～150nm）,and the particle size with the largest content was about 137.0nm,accounting for 99.4%,and the concentration was 4.1×10⁶.The results of WB showed that CD81 was positive,which was consistent with the description of related literature.3.Effect of ADMSCs-Exo on intimal hyperplasia of carotid artery balloon injury in rats3.1The results of hematoxylin-eosin stainingshowed that the common carotid arteries in the control group were observed at 14 and 28 days after operation.The vascular structure of the common carotid artery was normal,the lumen was normal,and no neointima was formed.The proliferation of vascular smooth muscle cells is active,and the intimal hyperplasia is large.Compared with the injury group,the exosomes group has a thicker wall,narrower lumen and intimal hyperplasia.The IA,MA,I/M ratios were calculated.The results showed that the I/M ratio of the exosomes group was lower than that of the injury group at 14 days after surgery,and the difference was statistically significant（0.18±0.01 vs 0.27±0.06;n=5;P<0.05）,there was also a significant statistical difference at 28 days（0.22±0.03 vs0.41±0.05;n=5;P<0.05）.3.2The results of Evans blue staining showed that at 14 days after operation:in the control group,the endothelium was intact,and no obvious endothelium was stained blue;in the injury group,large area of irregular lamellar endothelium was stained blue;in the exosomes group,some of the more regular endothelium was also stained;and compared with the control group,the ratio of endothelialized area/total blood vessel area in the injury group was significantly reduced（75.41±4.29%vs 97.49±0.98%;n=5;P<0.05）,while the exosomes group could improve the carotid artery after balloon injury endothelialization,compared with the injury group,the ratio of endothelialized area/total vascular area in the exosomes group increased significantly（86.07±2.94%vs 75.41±4.29%;n=5;P<0.05）.Conclusions:1.ADMSCs with multipotential differentiation could be obtained from adipose tissue;2.Exo-spin ^TMmethod can extract and purify the exosomes of ADMSCs cells;3.ADMSCs-Exo can inhibit intimal hyperplasia and stenosis after carotid artery injury in SD rats;4.ADMSCs-Exo can promote the endothelialization of injured carotid artery in SD rats.