| Objective:1.To investigate the effects of hUCMSC-MVs on FLS in patients with RA.2.To investigate whether hUCMSC-MVs can affect the secretion of cytokines of RAFLS by regulating miR-19a/TLR2 pathway.Methods:1.Isolation,culture and identification of RAFLS:The synovial tissue of 3 RA patients was collected.RAFLS was isolated and cultured by enzyme digestion.The third generation RAFLS was used for phenotypic identification.The surface antigens CD14and CD68,CD90 were detected by flow cytometry;2.Acquisition and identification of hUCMSC-MVs:hUCMSC-MVs were obtained by differential centrifugation in the early stage of the research group,and hUCMSC-MVs were successfully identified by electron microscopy and BCA[23].3.Experimental group:hUCMSC-MVs were mixed with RAFLS in vitro and divided into 5 groups:RAFLS control group,Bacterial lipoproteinlipoprotein?BLP?group?final concentration 0.4ug/ml?,hUCMSC-MVs low concentration group?10?g?./ml),hUCMSC-MVs medium concentration group?30?g/ml?,hUCMSC-MVs high concentration group?60?g/ml?.Repeat 3 times for each experiment.4.Treatment of experimental group:RAFLS control group:only DMEM complete culture solution was added without any stimulation;BLP group:BLP solution with final concentration of 0.4 ug/ml was intervened in RAFLS for 24 hours;hUCMSC-MVs low concentration group:after adding hUCMSC-MVs?10ug/ml?for 6h,BLP?0.4 ug/ml?was added and mixed for 24h;hUCMSC-MVs medium concentration group:after adding hUCMSC-MVs?30ug/ml?for 6h,BLP?0.4 ug/ml?was added and mixed for 24h;hUCMSC-MVs high concentration group:After adding hUCMSC-MVs?60ug/ml?for 6h,BLP?0.4 ug/ml?was added and mixed for 24h.5.Detection indicators:?1?CCK-8 method to detect cell proliferation;?2?RT-PCR detection of miR-19a,TLR2,IL-6,MMP-3 mRNA expression levels;?3?ELISA was used to detect the protein expression levels of IL-6 and MMP-3 in the cell culture supernatant;the data were collected for statistical analysis.Results:1.Isolation,culture and identification of RAFLSFlow cytometry was performed on RAFLS with a good growth state and a degree of fusion of 80-90%in P3 generation.The positive rate of CD90 on the cell surface was up to 98%,and CD14 and CD68?-?were consistent with RAFLS characterization.2.The effect of hUCMSC-MVs on the proliferation of RAFLS?1?Compared with the control group,the BLP group promoted the proliferation of RAFLS,and the difference was statistically significant?P<0.05?;?2?Compared with the BLP group,each hUCMSC-MVs group inhibited the proliferation of RAFLS?P<0.05?;?3?In the inhibition of RAFLS proliferation,the hUCMSC-MVs group?10ug/ml?was superior to the hUCMSC-MVs group?60ug/ml?,and the difference was statistically significant?P<0.05?.The proliferation of hUCMSC-MVs group?30ug/ml?was slightly weaker than that of hUCMSC-MVs group?10ug/ml?,and the difference was not statistically significant?P>0.05?.3.The effect of hUCMSC-MVs on the secretion of cytokines of RAFLs via miR-19a/TLR2 pathway?1?Changes in miR-19a gene expression level:Compared with the control group,miR-19a mRNA expression level was down-regulated in BLP group?P<0.05?;compared with BLP group,each hUCMSC-MVs group up-regulated miR-19a mRNA expression level?P<0.05?;hUCMSC-MVs?10ug/ml?were superior to hUCMSC-MVs?30ug/ml?and hUCMSC-MVs?60ug/ml?,the difference was statistically significant?P<0.05?;hUCMSC-MVs?30ug/ml?were superior to hUCMSC-MVs?60ug/ml?,and the difference was not statistically significant?P>0.05?.?2?Changes in TLR2 gene expression levels:Compared with the control group,the expression of TLR2 mRNA was significantly up-regulated in the BLP group?P<0.05?.Compared with the BLP group,the expression of TLR2 mRNA was down-regulated in each hUCMSC-MVs group?P<0.05?;hUCMSC-MV?10ug/ml?and hUCMSC-MV?30ug/ml?were better than hUCMSC-MV?60ug/ml?,the difference was statistically significant?P<0.05?.For the down-regulation of TLR2,hUCMSC-MV?30ug/ml?was slightly weaker than hUCMSC-MV?30ug/ml?,and the difference was not statistically significant?P>0.05?.?3?Changes in IL-6 gene and protein expression levels:Compared with the control group,the protein and gene expression levels of IL-6 in BLP group were significantly up-regulated?P<0.05?.Compared with BLP group,IL-6 mRNA and protein expression were down-regulated in hUCMSC-MVs group,the difference was statistically significant?P<0.05?.hUCMSC-MVs?10ug/ml?and hUCMSC-MVs?30ug/ml?were superior to hUCMSC-MVs?60ug/ml?in the down-regulation of IL-6 mRNA,the difference was statistically significant?P<0.05?;hUCMSC-MVs?30ug/ml?was slightly weaker than hUCMSC-MVs?10ug/ml?,the difference was not statistically significant?P>0.05?.In the down-regulation of IL-6 protein,hUCMSC-MVs?10ug/ml?was superior to hUCMSC-MVs?30ug/ml?,the difference was statistically significant?P<0.05?;hUCMSC-MVs?30ug/ml?was better than hUCMSC-MVs?60ug/ml??P<0.05?.?4?Changes in MMP3 gene and protein expression levels:Compared with the control group,the protein and gene expression levels of MMP3 in BLP group were significantly up-regulated?P<0.05?;Compared with the BLP group,the expression of MMP3 mRNA and protein was down-regulated in each hUCMSC-MVs group,and the difference was statistically significant?P<0.05?.In the down-regulation of MMP3 mRNA,hUCMSC-MVs?10ug/ml?wassuperiortohUCMSC-MVs?30ug/ml?and hUCMSC-MVs?60ug/ml?,and the difference was statistically significant?P<0.05?.In the down-regulation of MMP3 protein,hUCMSC-MVs?10ug/ml?was superior to hUCMSC-MVs?60ug/ml?,and the difference was statistically significant?P<0.05?.?5?Correlation analysis between TLR2 and IL-6 and MMP3:There was a high positive correlation between TLR2 mRNA and IL-6 mRNA?r=0.954,P<0.001?,and a high positive correlation with MMP3 mRNA?r=0.983,P<0.001?.There was a moderate positive correlation with IL-6 protein expression?r=0.716,P<0.01?,and a moderate positive correlation with MMP3 protein expression?r=0.757,P<0.01?.Conclusions:1.hUCMSC-MVs may treat RA by inhibiting the proliferation of RAFLS;2.hUCMSC-MVs may attenuate the inflammation of RA by modulating the miR-19a/TLR2 pathway. |
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