Construction Of Gout Susceptibility Gene RFX3 Conditioned Knockout Mice And Its Regulation Mechanism Of Inflammation

Objective: The previous study of our group found that RFX3 is a gout susceptibility gene in Chinese Han population,but the mechanism of its involvement in gout inflammatory process is unclear.In this study,a monocyte RFX3 conditional knockout mouse model was constructed,and the mouse model and primary bone marrow mononuclear cells were used to study the regulatory effect and mechanism of RFX3 on inflammatory cells and inflammatory factors in the pathogenesis of gout.Methods: 1.Based on the principle of homologous recombination,the RFX3 gene was modified by flox to obtain flox mice with conditional knockout of RFX3 gene by homologous recombination of fertilized eggs,and the flox mice were mat ched with Lyz2-Cre tool mice to obtain selective myeloid monocyte specific RFX3 knockout mice(KO mice)and control mice(WT mice).Mouse genotypes were identified by DNA,PCR method,bone marrow mononuclear cells were isolated and cultured,and the expression level of RFX3 was detected by immunofluorescence staining and Western blot method after 7 days of differentiation in the supernatant of L929 cells.2.The blood biochemistry and blood routine of KO and control mice were detected to determine the effect of conditional knockout of RFX3 gene on the phenotype of mice.3.The mouse model of peritonitis induced by MSU was established.4 hours later,the peritoneal lavage fluid of mice was collected,and the cells in the lavage fluid were isolated.The cells in the lavage fluid were blocked by Fc bl ocker,and incubated with ly6 G antibody(FITC labeled),CD3 antibody(Percp/cy5.5 labeled),F4/80 antibody(APC labeled),CD11 b antibody(PE labeled)and CD45 antibody(PB labeled).The numbers of monocytes / macrophages(CD45+F4/8 0+CD11b+),neutrophils(CD45+ly6G+CD11b+)and lymphoid T cells(CD45+CD3+)were detected and analyzed by flow cytometry.The cells in the peritoneal lavage fluid were collected,and the RNA,q RT-PCR was extracted to detect the m RNA expression of inflammatory factors such as IL-1β and IL-6,TGF-β,and the secretion level of IL-1β in the supernatant of the lavage fluid was detected by ELISA.4.Mouse bone marrow monocytes were isolated and cultured,and the supernatant of L929 cells was induced into monocytes / macrophages.The level of cell differentiation was detected by flow cytometry,and the cell migration ability was detected by cell scratch and Transwell test to determine the effect of RFX3 on the function of mouse monocytes.5.The effect of RFX3 on macrophage cytoskeleton was observed by phalloidine staining,and the mechanism of RFX3 regulating monocyte / macrophage migration and inflammation was studied by proteomics.Results: 1.Using the method of homologous recombination of fertilized eggs,w e successfully constructed RFX3 gene conditional knockout mice.The results of gene identification showed that flox and Cre bands were expressed in KO mice,and the expression level of RFX3 protein in monocytes of KO mice was significantly decreased by Westernblot and immunofluorescence staining.2.There was no significant difference in blood biochemistry and blood routine indexes between KO mice and the control group.3.In this study,we successfully established a mouse model of peritonitis induced by MSU.The results of flow cytometry detection and analysis of inflammatory cells in peritoneal lavage fluid of this model showed that the number of macrophages in RFX3 knockout mice was significantly higher than that in control mice,but there was no significant change in the number of neutrophiland lymphoid T cells.The results of m RNA detection showed that the expression levels of IL-1β,IL-6 and TGF-β in peritoneal lavage fluid of KO mice were no significantly lower than those of WT mice,while the expres sion levels of IL-1β in peritoneal lavage fluid of KO mice were significantly lower than those of WT mice.It is suggested that RFX3 may be involved in the regulation of monocyte / macrophage migration.4.The results of the study on the differentiation l evel of monocytes / macrophages showed that RFX3 knockout had no significant effect on the differentiation of bone marrow monocytes,but the cell scratch test showed that the migration ability of bone marrow monocytes in RFX3 knockout mice was significantly increased.The results of cell Transwell assay showed that the migrationumber of bone marrow monocytes in RFX3 knockout mice was significantly higherthan that in the control group(1052.27 ±56.88 / cm3 vs.824.6 ±45.71 / cm3).The results show that RFX3 knockout promotes the migration ability of monocytes / macrophages;5.The results of cytoskeleton staining sho wed that the adhesion area of bone marrow mononuclear cells and the number of foot corpuscles in KO mice were significantly increased,and proteomic studies showed that actin cytoskeleton pathway was closely related to gout.The above results suggest that RFX3 knockout may lead to changes in the cytoskeleton structure of monocytes / macrophages,which may be an important mechanism for the enhancement of migration ability of monocytes / macrophages.Conclusion: In this study,the model of monocyte RFX3 conditional knockout mice was successfully constructed,and gene knockout had no significant effect on the phenotype of mice,while RFX3 gene knockout could significantly reduce the level of gout inflammation,change the cytoskeleton structure of monocytes / ma crophages and promote monocyte / macrophage migration,which may be the key mechanism of gout inflammation regulation.