| Objective:The aims of this study are evaluating the effect of hUCMSC-MVs on the exp ression of chemokine CCL2,CXCL10 and CXCL12 in synovium and spleen of CI A rats,and exploring the mechanism of hUCMSCs-MVs affecting RA chemokines.Methods:(1)Microvesicles derived from Human Umbilical Cord Mesenchymal Stem Cells(hUCMSCs)was cultured in vitro,then hUCMSC-MVs are separated using a differential centrifugation methods..(2)Establishing The collagen induced arthritis(CIA)rat model:Using freund’s complete adjuvant and type II collagen emulsion immune the Wistar rats twice,then set up the the CIA model.The model observation target including the symptoms of rats(general view,arthritis index,joint swelling degree),imaging and pathological evaluation of joint.(3)Experiment group:CIA rats were randomly divided into CIA control group,MTX intervention group,hUCMSCs intervention group,hUCMSCs-MVs low concentration group,hUCMSCs-MVs high concentration group,and a normal control group was set up,6 rats in each group.(4)Intervention method:The hUCMSCs group:Rat tail vein injection,the dose was2×10~6 hUCMSCs/Kg;The hUCMSCs-MVs group:Rat tail vein injection,the dose of hUCMSCs-MVs low,high concentration groups was 30μg/100μL,90μg/100μL saline.MTX group:Intraperitoneal injection of methotrexate with 0.9 mg/kg,once a week,a total of three weeks.CIA group:Injection of equal volume of saline by the same method.(5)Test index:the measurement of the joint swelling degree,evaluation of arthritis index and imaging scores were used to observe the intervention efficacy of hUCMSC-MVs on CIA rats.The expression of CCL2 and CXCL10 in the serum were detected by Aimplex and FCAP Array software(V3.0)derived data,the expression of CXCL12 in the serum were detected by enzyme-linked immunosorbent assay(ELISA),Immunohistochemical technique was used to detect the expression of chemokines in the ankle joint.After modeling 42d,the synovial membrane and spleen RNA were extracted.The levels of CCL2,CXCL10 and CXCL12 mRNA in each group were measured by RT-PCR method.Results:1.Effects of h UCMSC-MVs intervention on the general situation of CIA rats:1.1 The hUCMSC-MVs effect on the body weight of CIA rats:compared with normal control group,the weight gain of CIA group significantly reduced(P<0.05).After intervention,the weight gain of CIA rats was better.And in 42d,the weight of hUCMSC-MVs high concentration group was significantly higher than that of CIA group(P<0.05).1.2 h UCMSC-MVs intervention on joint symptoms in CIA rats:compared with normal control group,the degree of foot swelling in CIA group increased significantly(P<0.05),as well as AI(P<0.05).After intervention,the degree of foot swelling and AI in CIA rats were declined.In 42d,the swelling degree and AI in MTX group were significantly reduced,the degree of foot swelling and AI were similar between CIA group,hUCMSCs group and hUCMSC-MVs group(P>0.05).1.3 The effect of hUCMSC-MVs on the ankle joint pathology in CIA rats:the articular cavity structure in normalcontrol rats was clear,and there were a small a mount of synovial hyperplasia and inflammatory cell infiltration.Synovial hyperplasi a was obvious,and a large number of inflammatory cell infiltrated in CIA rats,car tilage destruction were founded in some CIA rats.The degree of synovial hyperpla sia and inflammatory cell infiltration were similar in MTX group,hUCMSCs group,hUCMSC-MVs low,high concentration group.2.The effects of hUCMSC-MVs intervention on the serum expression of chemokines of CIA rats:2.1 Effect of h UCMSC-MVs on the serum CXCL10 of CIA rats:the level of serum CXCL10 in the group CIA was higher than that in the normal control group,but there was no statistical difference(P>0.05),and the serum CXCL10 level of the intervention group was close,and there was no significant difference between each intervention group and the CIA group(P>0.05).2.2 Effect of h UCMSC-MVs on the serum CXCL12 of CIA rats:the level of serum CXCL12 in the CIA group was higher than that in the normal control group,MTX group,hUCMSCs group and low,high concentration hUCMSC-MVs group(P<0.05).The level of serum CXCL12 in MTX group was equal to hUCMSCs group,and had no statistical difference with the low,high concentration hUCMSC-MVs group(P>0.05).The serum CXCL12 levels in different concentration hUCMSC-MVs groups were similar(P>0.05).2.3 The effect of hUCMSC-MVs on serum CCL2 in CIA rats:the level of serum CCL2in the CIA group was higher than that in normal control group,MTX group,hUCMSCs group and low,high concentration hUCMSC-MVs group(P<0.05).The serum CCL2level in MTX group was equal to that of the group,hUCMSCs group,and had no statistical difference with the low,high concentration hUCMSC-MVs group(P>0.05).The serum CCL2 levels in different concentration hUCMSC-MVs groups were similar(P>0.05).3.The effect of hUCMSC-MVs on the chemokine mRNA in the spleen of CIA rats:3.1 Effects of h UCMSC-MVs on spleen CXCL10 mRNA:CXCL10 mRNA conte nt of spleen in CIA group was higher than the normal control group,MTX group,low and high concentration of hUCMSC-MVs group(P<0.05),but similar to hUC MSCs group(P>0.05);CXCL10 mRNA content in high and low concentration of h UCMSC-MVs group were similar(P>0.05),and had no difference with hUCMSCs group(P>0.05).3.2 Effects of h UCMSC-MVs on spleen CXCL12 mRNA:CXCL12 mRNA conte nt in CIA group was significantly higher than other groups(P<0.05);The mRNA e xpression in MTX group was similar to hUCMSCs group and low concentration of hUCMSC-MVs group(P>0.05);CXCL12 mRNA level in high concentration of h UCMSC-MVs group was lower than the low concentration group(P<0.05).3.3 Effect of hUCMSC-MVs on spleen CCL2 mRNA:The content of CCL2 mR NA of spleen in CIA group is higher than that of MTX group,hUCMSCs group,low and high concentration of hUCMSC-MVs group(P<0.05).The mRNA content in hUCMSCs group is similar to that in MTX group(P>0.05).Compared with hUC MSCs group,there was no significant difference in low and high of hUCMSC-MV s concentration group(P>0.05).The CCL2 mRNA content in high and low concent ration of hUCMSC-MVs group was equal(P>0.05).4.The effect of hUCMSC-MVs on the expression of chemokines in synovium membrane of CIA rats:4.1 The effect of hUCMSC-MVs on the expression of CXCL10 in synovium m embrane of CIA rats:(1)Effects of hUCMSC-MVs on synovial CXCL10 protein:The synovial tissue o f CIA rats was proliferated and the expression of CXCL10 was strong positive.Th e CXCL10 in the synovial membrane of the normal group was weak positive or n egative,and the CXCL10 in the synovium of the intervention group was weakly p ositive.The semi quantitative analysis showed that the expression of CXCL10 in t he synovial membrane of the CIA group was significantly higher than that of the normal control group(P<0.05),and the expression of CXCL10 in the intervention groups was significantly lower than that in the CIA group(P<0.05).Compared wit h MTX group,the CXCL10 expression in the synovium of the low concentration hUCMSC-MVs group was significantly higher(P<0.05),Compared with hUCMSCs group,the expression of CXCL10 in the synovial membrane of the low concentra tion hUCMSC-MVs group was significantly increased(P<0.05).The CXCL10 expre ssion in the hUCMSCs group was close to that of the MTX group(P>0.05),and t he CXCL10 level in the synovium of the low concentration group was higher than that in high concentration group(P<0.05).(2)Effects of h UCMSC-MVs on synovial CXCL10 mRNA:Compared with CIA group,the CXCL10 mRNA content of synovial was lower significantly in normal c ontrol group,MTX group,hUCMSCs group,low concentration of hUCMSC-MVs g roup,high concentration of hUCMSC-MVs group(P<0.05);CXCL10 mRNA conten t in MSCs group is lower than the low and high concentration of hUCMSC-MVs group(P<0.05);The expression of CXCL10 mRNA in high concentration of h UC MSC-MVs group was lower than that in the low concentration group,but no signi ficant difference(P>0.05).4.2 The effect of hUCMSC-MVs on the expression of CXCL12 in synovium m embrane of CIA rats:(1)Effects of h UCMSC-MVs on synovial CXCL12 protein:The synovial tissue of CIA rats was proliferated and the expression of CXCL12 was strong positive.T he CXCL12 in the synovial membrane of the normal group was weak positive or negative,and the CXCL12 in the synovium of the intervention groups was weakly positive.By semi quantitative analysis,the expression of CXCL12 in the synovial membrane of CIA group was significantly higher than that in the normal control group(P<0.05),and the expression of CXCL12 in the intervention groups was sign ificantly lower than that in the CIA group(P<0.05).The CXCL12 expression in t he hUCMSCs group was close to that of MTX group(P>0.05).Compared with M TX group,the CXCL12 expression in the synovium of the low concentration hUC MSC-MVs group was significantly higher(P<0.05).Compared with hUCMSCs grou p,the expression of CXCL12 in the synovial membrane of the low concentration h UCMSC-MVs group was significantly increased(P<0.05).and the CXCL10 level o f the low concentration group was higher than that in the high concentration group(P<0.05).(2)Effects of h UCMSC-MVs on synovial CXCL12 mRNA:Synovial CXCL12 m RNA in CIA group was higher than that of other groups(P<0.05);the mRNA cont ent in hUCMSCs group was similar to MTX group(P>0.05),and that was lower t han the low concentration of hUCMSC-MVs group and high concentration of hUC MSC-MVs group(P<0.05);CXCL12 mRNA content in hUCMSCs group was simil ar to high and low concentration of hUCMSC-MVs group(P>0.05);There was no significant difference between the low and high concentration of hUCMSC-MVs g roup(P>0.05).4.3 The effect of hUCMSC-MVs on the expression of CCL2 in synovium mem brane of CIA rats:(1)Effects of hUCMSC-MVs on synovial CCL2 protein:The expression of CCL2in synovial tissue of CIA rats was strong positive.The CCL2 in the synovial me mbrane of the normal group was weak positive or negative,and the CCL2 in the synovium of the intervention groups was weakly positive.The semi quantitative an alysis showed that the expression of CCL2 in the synovial membrane of the CIA group was significantly higher than that of the normal control group and each inter vention groups(P<0.05).The CCL2 expression in the hUCMSCs group was close t o that of the MTX group(P>0.05),The CCL2 in the synovium of the low concen tration hUCMSC-MVs group was significantly higher than MTX group and hUCM SCs group(P<0.05),as well as the high concentration hUCMSC-MVs group(P<0.05).(2)Effects of hUCMSC-MVs on synovial CCL2 mRNA:CCL2 m RNA of synoviu m in CIA group were higher than MTX group,hUCMSCs group,low concentratio n of hUCMSC-MVs group and high concentration of hUCMSC-MVs group(P<0.05);compared with the low concentration of hUCMSC-MVs group,CCL2 mRNA c ontent in MTX group,hUCMSCs group,and hUCMSC-MVs high concentration gr oup were lower(P<0.05);CCL2 mRNA in high concentration of hUCMSC-MVs group was as much as MTX group and hUCMSCs group(P>0.05).Conclusion:1.hUCMSC-MVs can alleviate the inflammatory reaction by inhibiting the expression of chemokine CCL2,CXCL10 and CXCL12 in CIA rats..2.The inhibitory effect of high concentration of hUCMSC-MVs on chemokines in CIA rats was similar to that of hUCMSCs and MTX.3.The inhibitory effect of high concentration of hUCMSC-MVs on the chemokines in synovial membrane was better than that of low concentration. |
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