| Background:The immune regulation disorder caused by epigenetic abnormalities is involved in the occurrence and development of rheumatoid arthritis(RA).Histone acetylation modification is a reversible epigenetic modification which is occured in histone lysine residues.Several studies have shown that it regulates and participates in the pathogenesis of RA through histone deacetylase 1(HDAC1)and HDAC2.Our group’s previous studies found that human umbilical cord mesenchymal stem cells derived microvesicles(hUCMSC-MVs)can improve the joint symptoms and images of Collagen-induced arthritis(CIA)rats.Academic performance,inhibition of synovial hyperplasia,down-regulation of pro-inflammatory factors and chemokine levels,and the effect is not weaker than maternal cells.Fibroblast-like synoviocytes(FLSs)are the core cells in RA pathology and play a key role in joint inflammation and bone destruction of RA.The effect of hUCMSC-MVs on the function of rheumatoid arthritis fibroblast-like synoviocytes(RA FLSs)and its mechanism of action are still unclear.Objective:To investigate the effects of hUCMSC-MVs on proliferation,apoptosis,migration and expression of HDAC1 and HDAC2 in RA FLSs,we would use the vitro experiments by coculturing hUCMSC-MVs with RA FLSs in this study.The genetic histone acetylation perspective provides an experimental basis for the treatment of RA with hUCMSC-MVs.Methods:1.Isolation and identification of hUCMSC-MVs: hUCMSCs were resuscitated,P3-P7 generation was taken after passage.Through the hUCMSCs’ "starvation" cultured for 24 hours,microvesicles are separated from mesenchymal stem cells conditioned supernatant using a differential centrifugation methods.Morphology was observed by transmission electron microscopy.Surface molecular markers were identified by Western Blot.2.Isolation,culture and identification of OA FLSs: OA FLSs were isolated and cultured in vitro by trypsinization,and their morphology was observed under an inverted microscope.The surface antigens CD90,CD14 and CD45 were determined by flow cytometry.3.hUCMSC-MVs were co-cultured with RA FLSs: the effects of proliferation,apoptosis,migration and expression of HDAC1 and HDAC2 on RA FLSs were observed.(1)Experimental group: A total of 4 groups were OA group,RA group,Trichostatin A(TSA)group and hUCMSC-MVs group.(2)Detection index:(1)CCK8 method to detect cell proliferation rate,flow cytometry to detect apoptosis rate,scratch test to detect cell migration;(2)Western Blot method to determine the expression of HDAC1 and HDAC2 in RA FLSs before and after hUCMSC-MVs intervention.Results:1.Isolation and identification of hUCMSC-MVs: Transmission electron microscopy showed that the products obtained by centrifugation were a kind of vesicular structure with different diameters ranging from 40 nm to 300 nm.Western Blot identified molecular surface markers,and CD63 and HSP70 were positive.2.Isolation and identification of OA FLSs: The cells were observed to have a vortex-like distribution under an inverted microscope,which was a long spindle-shaped,spindle-like appearance.Flow cytometry identified molecular surface markers,CD 90 positive expression,CD14,CD45 negative expression.3.In vitro co-culture of hUCMSC-MVs with RA FLSs:(1)The effect of hUCMSC-MVs on the proliferation function of RAFLSs:hUCMSC-MVs group concentration was 10ug/ml,50ug/ml,100ug/ml,150ug/ml,200ug/ml.1)Compared with the RA control group,the proliferation rate of FLSs was increased in the hUCMSC-MVs group at a concentration of 10 ug/ml,50 ug/ml,100 ug/ml,and the proliferation of FLSs in the hUCMSC-MVs group and the TSA group at a concentration of 150 ug/ml,200 ug/ml.The rate decreased and the difference was statistically significant(P <0.001).2)There was no significant difference in the proliferation rate of FLSs between the150 ug/ml hUCMSC-MVs group and the 200 ug/ml hUCMSC-MVs group(P>0.05).3)compared with the TSA group,150ug/ml hUCMSC-The proliferation rate of FLSs in MVs group and 200ug/ml hUCMSC-MVs group increased,but there was no significant difference(P>0.05).(2)The effect of hUCMSC-MVs on the apoptosis function of RA FLSs: The apoptosis rate of FLSs in hUCMSC-MVs group and TSA group was higher than that in RA control group(P<0.05).Compared with the TSA group,the apoptotic rate of FLSs in the hUCMSC-MVs group was increased,but there was no significant difference(P>0.05).(3)The effect of hUCMSC-MVs on the migration function of RA FLSs: At 12 h and24h,the migration rate of FLSs in hUCMSC-MVs intervention group and TSA group was lower than that in RA control group(P<0.05).The migration rate of FLSs in hUCMSC-MVs group was higher than that in TSA group,but there was no significant difference(P>0.05).(4)The effect of hUCMSC-MVs on the expression of HDAC1 and HDAC2 in RAFLSs: The expression of HDAC1 and HDAC2 in RA FLSs group were higher than those in OA FLSs group(P<0.05).Compared with RA FLSs group,hUCMSC The expression of HDAC1 protein was decreased in the-MVs group(P<0.05),and the effectwas not weaker than that in the TSA group.Conclusions:hUCMSC-MVs can inhibit the proliferation and migration of RA FLSs and promote their apoptosis.HDAC1 and HDAC2 are involved in the pathogenesis of RA.hUCMSC-MVs can down-regulate the expression of HDAC1 in RA FLSs and inhibit the pathogenesis of RA from the perspective of histone acetylation. |
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