Effect Of Human Umbilical Cord Mesenchymal Stem Cells Derived Microvesicle On Peripheral Blood Lymphocytes And Inflammatory Factors In Patients With Rheumatoid Arthritis

Background:Rheumatoid arthritis(RA)is a chronic,systemic,inflammatory autoimmune disease.The pathological changes in addition to synovial hyperplasia and pannus formation and destruction of bone and cartilage tissue,the most fundamental is there is a disorder of the immune function.Large number of studies confirmed that Treg/Th17 cell’s imbalance in the occurrence and development of RA plays a crucial role.Previous studies have showed that mesenchymal stem cells(MSCs)is a pluripotent stem cell,which has a strong ability to proliferate and a number of differentiation potential.It is important that the immune regulation function can play a certain therapeutic effect to RA.However,The source of MSCs is limited.In recent years,Some studies found mesenchymal stem cells conditioned supernatant extracted microvesicles(MSC-MVs)also have immune regulatory effect.It may have better prospects for treatment in autoimmune diseases.Objective:MSCs are separated from human umbilical cord by tissue block culture method,which is purifed by passage cells.We are observing the osteogenic and adipogenic differentiation ability.Then using flow cytometry detects the phenotype.Through the hUC-MSCs’ "hunger" cultured for 24 hours,microvesicles are separated from mesenchymal stem cells conditioned supernatant using a differential centrifugation methods.In vitro hUC-MSCs and hUCMSC-MVs are mixed cultured with RA patients’ PBMC for three days.Then using flow cytometry detects Th17cells’ and Treg cells’ expression levels and some inflammatory factors’ changes.Methods:1.Isolation and identification of hUC-MSCs and hUCMSC-MVs(1)Isolation and identification of hUC-MSCs :In vitro using tissue culture method sepatates hUC-MSCs.Flow cytometry technique detects surface antigen CD29,CD44,CD105,CD73,CD90,CD166,CD34 and CD45.Under the specific conditions,hUC-MSCs’ ability of osteoblast and adipogenic differentiation potential were identified.(2)Isolation and identification of hUCMSC-MVs: Through the "hunger" cultured for24 hours,microvesicles are separated from mesenchymal stem cells conditioned supernatant using a differential centrifugation methods.The morphology was observed by transmission electron microscope,and BCA method was used to determine the protein content in the microplate reader.2.hUCMSC-MVs in vitro mixed with RA in patients with PBMC(1)Grouping: A total of five groups.The treatment groups contain hUCMSC-MVs low,medium and high concentration group,which mean that the quantity of hUCMSC-MVs is different.Meanwhile,there are blank control group and psitive control group.The latter is equal to hUCMSCs treatment group.(2)Index detection:Th17 cells and Treg cells’ expression levels and some inflammatory factors’ changes is detected by flow cytometry software(V3.0)derived data,and collect data for statistical analysis.Results:1.Isolation and identification of hUC-MSCs and hUCMSC-MVs(1)Isolation and identification of hUC-MSCs :Cells were positive for CD29,CD44,CD73,CD90,CD105 and CD166,and negative for CD34,CD45.Ability of mesenchymalstem cells induced to differentiate into osteoblasts and adipocytes.(2)Isolation and identification of hUCMSC-MVs: By transmission electron microscopy,the results show that the product is a kind of vesicle like structure,which size is between 40 nm and 300 nm diameter.Protein concentration is about 2.05μg/μl.2.hUCMSC-MVs in vitro mixed with RA in patients with PBMC(1)Grouping: Compared with the blank control group,hUC-MSCs group’s Th17 and IL-17A、IL-2、IL-1a、TNF-α can be decreased significantly.Moreover,Treg cells and TGF-β 、 IL-10 were increased significantly.The difference is statistically significant(P<0.05).(2)Comparison of low,middle and high concentration hUCMSC-MVs group,high concentration group can significantly reduce the expression of Th7 cells and IL-17A、IL-1a、TNF-α and increase the expression of Treg cells and TGF-β、IL-10,the difference was statistically significant(P<0.05).(3)Compared with the hUC-MSCs group,hUCMSC-MVs’ high concentration group in reducing expression of Th7 cells andIL-17 A 、 IL-1a 、 TNF-α,and increasing the expression of Treg cells and TGF-β、IL-10 function has no significant difference(P>0.05).In other words,Therapeutic effect of hUC-MSCs and hUCMSC-MVs’ high concentration group are equivalent(P>0.05).Conclusions:hUC-MSCs and different concentration gradient of hUCMSC-MVs were mixed culture with RA patients’ PBMC for 3 dsays in vitro.Study found two treatments can be reduced Th17 and IL-17A、IL-2、IL-1a、TNF-α levels,and upregulation of Treg and TGF-β 、 IL-10 level.What’s more,the hUC-MSCs’ effect is equal to the high concentration group of hUCMSC-MVs.