Study On The Role Of Synovial Mesenchymal Stem Cells And Their Microenvironment In The Development Of CIA Mice

Background:Rheumatoid arthritis(RA)is a chronic autoimmune disease characterized by pannus formation and infiltration of immune cells,which can lead to joint deformity and loss of function in severe cases.At present,the pathogenesis of RA has not been fully clarified.Some RA patients cannot get improvement under the existing treatment methods.In physiological state,synovial mesenchymal stem cells(SM-MSC)can regulate immunity,promote chondrogenesis,and maintain joint stability.The application of SM-MSC is expected to be a new method for RA treatment.MSC can have different characteristics in different microenvironments.Therefore,the role of SM-MSC and their microenvironment in RA has attracted more and more attention.The change of SM-MSC and the microenvironment in different stages of RA need to be discussed.Objective:1.Collagen induced arthritis(CIA)mice,classic RA animal model,are used to compare the characteristics of SM-MSC and their microenvironment in different stages of disease.2.To explore the effects of SM-MSC and microenvironment changes in diseased joints from the perspective of animal experiments,and provide experimental and theoretical basis for clarifying the pathophysiological changes in the pathogenesis of RA.Methods:1.Inducing collagen-induced arthritis(CIA)in DBA1/J mice: during the induction of the experimental group,joint thickness were measured regularly to evaluate their changes.The mice were divided into healthy control group(no redness and swelling),early CIA group(1-2 days after onset),mid CIA group(about 15 days after onset)and late CIA group(about 60 days after onset)according to the onset time of redness and swell.X ray and HE staining were performed on some mice.Destruction of bone and cartilage,synovial hyperplasia and inflammatory cell infiltration were evaluated.2.The sequencing of synovium: by using bioinformatics methods such as heat map,volcano map,GO-KEGG enrichment analysis,we compared the differences of sequencing results in different disease stages of mice,and selected genes with significant differences.The genes related to inflammation,stem cells,bone and cartilage destruction,cell matrix and immune cells were selected from the gene.3.SM-MSC extraction and identification: extract the synovium from the knee joint of the mouse’s hind limb,partially digest with enzyme and culture the primary cells,and stain the SM-MSC-specific cell surface marker(CD44)to identify whether the extracted cells are SM-MSC.4.Staining,cell viability and migration of SM-MSC: Cell viability and migration were evaluated in different groups by CCK8 and transwell.The results of different groups of mice were compared.4.SM-MSC injection: SM-MSC extracted from mid CIA group were injected into the ankles of healthy control group.SM-MSC extracted from the control group were injected into the ankles of experimental group.Joint thickness were measured regularly.Some of the joints were examined by HE staining.Results:1.Characteristics of SM-MSC and their microenvironment in different disease stages:(1)Joint thickness: The mid CIA group have the strongest joint redness and swelling.Joint stiffness and limited activity were found in the late CIA group.(2)Joint pathology: mid CIA group has the most severe inflammatory cell infiltration and synovial tissue hyperplasia.(3)X-ray: mid and late CIA group has the most severe bone destruction.Joint fusion and stiffness were observed in the late stage group of CIA.2.Transcriptome sequencing of synovial tissue: Compared with the healthy control group,MMP13,NELL1,SOX5,CCL3,THY1,ACAN,GREM1,CCN6,COL2A1 and other genes had high expression in experimental group(|log FC|>1.5 and P<0.05);CDH19,CLDN4 and other genes had low expression in experimental group(|log FC|>1.5 and P<0.05).By using GO-KEGG analysis,we found that these different genes were mainly enriched in several important signal pathways,such as leukocyte migration,intercellular junction,extracellular matrix structure,cartilage structure,cytokine activity,chemokines,metallopeptidase activity,etc.3.SM-MSC extraction and identification: The extracted cells expressed SM-MSCspecific surface marker CD44.4.SM-MSC stem gene ecpression,cell viability and cell migration ability of different group: immunofluorescence staining showed that the expression of transcription factors,including SOX2 and OCT4,were higher in experimental group.CIA mid stage group had the highest expression.Results of cell viability showed that the SM-MSC activity of the experimental group was significantly higher than that of the healthy control group.There was no significant difference in cell migration ability among different groups.5.Effect of SM-MSC injection: SM-MSC from inflammatory microenvironment do not change the joint outcome of healthy mice.SM-MSC from healthy microenvironment can improve the symptoms of joint redness and swelling in mice.Conclusion:1.CIA mice at different disease stages have different joint characteristics.The degree of joint damage gradually increase.Early intervention may effectively improve the symptoms and reduce the progress of RA.2.Compared with the control group,CIA groups had significantly up-regulated genes in inflammatory factors,extracellular matrix,bone destruction,MSC-specific markers and other aspects.Among the common up-regulated genes in different experimental groups,mid CIA group had the highest expression.3.SM-MSC exist in synovia of DBA1/J mouse.4.Compared with the control group,the expression of SM-MSC stem gene in CIA mice was more active.The cell viability was stronger in experimental groups,especially in mid stage CIA group.There was no significant difference in SM-MSC migration ability among different groups of mice.5.SM-MSC from the inflammatory microenvironment have no significant effect on healthy joints,while SM-MSC from the healthy microenvironment can improve the swelling of joints in CIA mice.