Protective Effect Of Human Umbilical Cord Mesenchymal Stem Cell-derived Exosomes On Rat Retinal Neurons In High Glucose Environment

Objective: To establish a retinal neuron model in a high glucose environment,coculture exosomes derived from human umbilical cord mesenchymal stem cells(h UCMSCs)with rat retinal neurons in a high glucose environment.To explore whether human umbilical cord mesenchymal stem cell-derived exosomes can exert the neuroprotective effects and possible neuroprotective mechanisms.Method: 1.Isolation and identification of h UCMSCs-EVs:The primary h UCMSCs were cultured to the 8th generation.h UCMSCs-EVs were isolated by ultracentrifugation.The diameter and structure of h UCMSCs-Evs were observed by transmission electron microscopy.The expression of surface marker proteins CD63 and CD90 in h UCMSCsEVs was detected by Western blot.2.ELISA was used to identify BDNF and its concentration in h UCMSCs-Evs.3.Establish a primary cultured rat retinal neuron high glucose model.4.According to the purpose of the experiment,the experimental cells were divided into 4 groups:(1)retinal neurons + Glu5.5mmol/L(normal control group),(2)retinal neurons + Glu35mmol/L(high glucose control group),(3)Retinal neurons + h UCMSCs-EVs(100 ng/ml)+ Glu 35 mmol/L(high glucose co-culture group),(4)retinal neurons + h UCMSCs-EVs(100 ng/ml)+ Glu 35 mmol/L + K252a(10 μg/ Ml)(high glucose co-culture group after BDNF inhibition).5.To detect the effects of h UCMSCs-EVs on the proliferation and apoptosis of high glucose retinal neurons(RGC): 1.The expression of BDNF receptor protein Trk B was observed by chemical immunofluorescence;The survival rate of retinal neurons in each group was determined by azozolium blue method(MTT).The apoptosis rate of retinal neurons in each group was detected by flow cytometry-Armexin/PI double staining.Results: 1.Successfully culture,isolate,extract and identify exosomes of h UCMSCs.2.Successfully extract and establish a rat retinal neuron model in a high glucose environment.3.Determination of BDNF content in exosomes of h UCMSCs by ELISA.4.After establishing rat retinal neuron model in high glucose environment,the cell number was detected by MTT colorimetry and the apoptosis rate of retinal neurons in each group was detected by flow cytometry-Armexin/PI double staining.Among them,the high glucose co-culture group and the high glucose control group and the high glucose co-culture group after BDNF inhibition had more cells,and the number of neuronal apoptosis was less,and there was significant statistical difference(P<0.05).5.After Trkb immunofluorescence staining,the expression of Trkb protein in the high glucose co-culture group was higher than that in the high glucose control group and the high glucose co-culture group after BDNF inhibition,the difference was statistically significant(P<0.05).Conclusion: 1.This study used exosomes extracted from h UCMSCs and found that in high glucose environment,BDNF can be promoted to promote the expression of Trek protein in retinal neurons and reduce the apoptosis of optic neurons in high glucose environment.Therefore,it can effectively protect retinal neuronal cells.2.This experiment further explains why h UCMSCs can protect retinal neurons in a high glucose environment and circumvent the risk of tumorigenesis and abnormal differentiation of h UCMSCs in therapeutic effects,and also effectively transfer BDNF to retinal neuronal cells.Stem cell treatment efficiency and utilization.