| 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（10μg）./ml),hUCMSC-MVs medium concentration group（30μg/ml）,hUCMSC-MVs high concentration group（60μg/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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