Human Umbilical Cord Mesenchymal Stem Cells-derived Exosomes Affect The Biological Traits Of Synovial Fibroblasts In Patients With Rheumatoid Arthritis Via Delivery Of MiRNA

Background:Rheumatoid arthritis is erosive and chronic symmetrical polyarthritis,and its pathological changes are joint synovial hyperplasia,cartilage and bone destruction.FLSs play a central role in the pathogenesis of RA.Abnormal expression of miRNA in RAFLSs promotes RA joint inflammation,synovial hyperplasia,and tissue destruction;MSC-Exos is a 30-150 nm membrane secretion system produced by MSCs,which not only retains the function of MSCs,but also has many unique advantages,such as precise and efficient delivery methods and structural advantages that are easy to store and control.The miRNA of MSC-Exos is the key molecule of the biological effect of MSC-Exos,and the directional transport of miRNA in organisms has extremely high therapeutic and transformational application prospects.Then whether hUCMSC-Exos can deliver miRNAs to RAFLSs and affect their biological properties remains to be verified by further studies.Objective:Whether hUCMSC-Exos can deliver RNA to RAFLSs;whether hUCMSC-Exos affects the proliferation and migration of RAFLSs by delivering miRNA.Methods:1.The uptake effect of RAFLSs on hUCMSC-Exos RNAPrimary hUCMSCs were isolated and cultured by tissue adherence method,surface markers were identified by flow cytometry,hUCMSC-Exos were obtained by differential centrifugation combined with ultrafiltration method,and further identified by electron microscope,nanoparticle tracking analysis,highly sensitive nanoparticle size analysis and BCA method The primary RAFLSs were isolated and cultured by trypsin digestion,and surface markers were identified by flow cytometry;the RNA labeled with hUCMSC-Exos,hUCMSC-Exos and RAFLSs were co-cultured for 24h,fixed and stained,and then placed in a fluorescence microscope for observation.2.Construction of hUCMSCs ^KD-Ago2Lentivirus GV493 was used to infect hUCMSCs to determine the optimal infection conditions,and then three lentiviruses GV493 containing different si RNA target sequences that could knock down Ago2 were used to infect hUCMSCs.RT-PCR and Westen blot were used to detect the expression of Ago2 in hUCMSCs,hUCMSCs ^KD-Ago2,hUCMSCs ^NC,and determine the target with the highest knockdown efficiency for the construction of hUCMSCs ^KD-Ago2.3.Detect the effect of miRNA in hUCMSCs-Exos on RAFLSsDifferential centrifugation combined with ultrafiltration was used to obtain Exos derived from hUCMSCs with Ago-2 gene knockdown,namely hUCMSCKD-Ago2-Exos,and Exos derived from hUCMSCs without Ago-2 gene knockdown,namely hUCMSCNC-Exos,using real-time label-free cell detection to study the effects of different concentrations of hUCMSC-Exos on the proliferation and migration of RAFLSs,determine the optimal intervention concentration,and further use a real-time label-free cell detector to compare the effects of hUCMSCKD-Ago2-Exos,hUCMSCNC-Exos and the blank control group on the proliferation and migration of RAFLSs.Results:Primary hUCMSCs were fusiform spindle-like,positive for CD105,CD73,and CD90,negative for CD45,CD34,and HLA-DR,in line with the characteristics of hUCMSCs.The morphology of primary RAFLSs was fibrous spindle-like,PDPN,CDH-11 positive,CD68 negative,in line with the characterization of RAFLSs;hUCMSC-Exos labeled RNA and RAFLSs were incubated with RAFLSs,and green fluorescent substances were gathered around the nucleus of RAFLSs stained in blue,and RAFLSs could ingest hUCMSC-Exos RNA.Lentivirus GV493 infects hUCMSCs with moderate difficulty,and the cells proliferate normally after infection.The optimal infection condition is adding infection enhancer Hi Trans G P MOI=20,and 3 lentiviruses carrying different target sequences can successfully infect hUCMSCs.RT-PCR and Western blot were used to detect the knockdown efficiency of Ago2 gene in infected hUCMSCs,and determine the viral target LVPSC85386-11 to infect hUCMSCs for experiments.It has been identified that hUCMSC-Exos,hUCMSC^KD-Ago2-Exos,and hUCMSC^NC-Exos are typical cup-holder-like and complete morphological membranous vesicles with a diameter of30～150nm,and the protein concentration is 1.384ug/ul,1.133ug/ul,1.038ug/ul.The intervention of hUCMSC^KD-Ago2-Exo and hUCMSC^NC-Exos in RAFLSs showed that the hUCMSC^KD-Ago2-Exos group and hUCMSC^NC-Exos group inhibited the proliferation and migration of RAFLSs at the same time point.The hUCMSC^NC-Exos group had the strongest effect,and the difference was statistically significant（p<0.01,P<0.05）;In the intervention group and the blank control group,the RAFLSs proliferation and migration cell index at different time points are statistically different（p<0.01,p<0.05）,and each group is overall There were statistical differences（p<0.01,p<0.05）.Conclusion:The uptake of hUCMSC-Exos RNA by RAFLSs may affect the proliferation and migration of RAFLSS through miRNA.