| Background: The increasing prevalence of diabetes has become the focus of global public health attention in the 21 st century.There are 463 million adults(20-79 years old)with diabetes in the world.Among them,diabetic ulcer is one of the most serious and costly complications of diabetes.To explore the effect of fibroblast exosomes from diabetic rats on wound healing,and find out the key target genes through miRNA sequencing,so as to find out new reasons of diabetic wound unhealed.Method: 30 SD rats of about 150 g were numbered and then randomly divided into experimental group and control group according to the random number table.Rats in the experimental group were injected with 55 mg/kg streptozotocin citrate buffer,while rats in the control group were injected with the same amount of citrate buffer.After 7 days,weight,blood glucose,water intake and urine volume of these rats were measured.The success index of the model was that the blood glucose was more than 16.7mmol/L,weight was significantly reduced compared with the control group,and the water intake and urine volume were significantly increased compared with the control group.The model was applied to the experiment after 12 weeks.Fibroblasts from the back skin of rats in the experimental group and the control group were extracted by tissue adherence method respectively.Cells were subcultured to P3 generation.P3 generation rat fibroblasts was cultured by complete culture medium combined with exosome-free serum and DMEM.Concentrating the collected cell supernatant with ultrafiltration tube,loading into ultracentrifugation tube,performing differential centrifugation to extract exosomes,and performing electron microscopy,Nanoparticle Tracking Analysis(NTA),and western-blot for identification.Using CCK-8 method to detect the effect of diabetic rat-derived fibroblast exosomes(Diabetes Exosome)on cell proliferation of human epidermal keratinocytes line(HEK-a)and human microvascular endothelial cell line(HMEC-1)compared with normal rat-derived fibroblast exosomes(Normal Exosome).Established rat back full-thickness skin wound model to observe the effect of diabetic rat-derived fibroblast exosomes and normal rat-derived fibroblast exosomes on rat wound healing compared with PBS control group.TotalRNA of fibroblast exosomes from diabetic rats and normal rats were extracted for miRNA.Then,differential expression analysis of known miRNA is carried out,and target gene information of the obtained differential miRNA is predicted.Functional annotation and enrichment analysis of target genes were carried out according to the prediction results.Result: 10 of the rats meet the criteria for successful modeling.The weight of the rats in the experimental group was significantly lower than that in the control group,and the blood glucose level,daily drinking water volume and urine volume were significantly higher than those in the control group.The primary fibroblasts were elongated and spindleshaped with clear and abundant cytoplasm.The typical exosome bilayer membrane structure can be seen by Transmission electron microscope.NTA suggested that the diameter of exosome was 50-200 nm,meet the requirements.Western-blot of exosomes suggested that positive indexes of exosomes,heat shock protein 90(HSP90)and heat shock protein 70(HSP70),were higher than those of cell control group,while negative indexes GM130 and β-tubulin of exosomes were not detected.CCK-8 test showed that after adding Diabetes Exosome and Normal Exosome,the absorbance of HEK-a and HMEC-1 at 450 nm was significantly higher than that of the control group.However,after adding Normal Exosome,the absorbance of HEK-a and HMEC-1 increased more at 450 nm.In the rat wound model,since the 7th day,compared with the control group,the proportion of wound surface in the Normal Exo group decreased significantly at the same time.Compared with the control group,the proportion of wound surface in Diabetes Exo group had no statistical difference at the same time.MiRNA sequencing results showed that the exosomes contained only a small number of miRNA,among which rno-mi R-99b-5p and rno-mi R-351-5p were significantly different,may be involved in tight intercellular connection,retrograde endogenous cannabinoid signal and caffeine metabolism.Conclusion: We have successfully extracted fibroblasts from diabetic rats and successfully extracted their exosomes.The content of HSP70 in fibroblasts derived from diabetic rats is lower than that of normal rats.CCK-8 test indicates that the ability of fibroblast exosomes from diabetic rats to promote proliferation of human keratinocyte cell lines and human microvascular endothelial cell lines is lower than that from normal rats.The results of rat wound model show that the ability of diabetic rat derived fibroblast exosomes to promote wound healing is lower than that of normal rat derived fibroblast exosomes.Sequencing of miRNA in rat fibroblast exosomes indicates that the down-regulation of rno-mi R-99b-5p and rno-mi R-351-5p expression in diabetic rat derived fibroblast exosomes may be the reason for the decrease of its ability to promote wound healing. |
