Effect Of Peripheral Blood-derived Mesenchymal Stem Cells On Macrophage And Th17/Treg-relevant Balance In Vitro

Objective:1.To investigate the difference in the expression of cytokines when blood-derived mesenchymal stem cells?PBMSCs?and bone marrow-derived mesenchymal stem cells?BMMSCs?were co-cultured with macrophages?M0?.2.To explore the Th17/Treg-relevant balance of peripheral blood-derived mesenchymal stem cel s in vitro.Method:Part one:The expression pattern of cytokines in mesenchymal stem cells and macrophages co-cultured system.1.Isolation,culture and identification of PBMSCs:Sprague Dawley?SD?rats,weighting around 100 g,were first ly intraperitoneally injected with G-CSF(100?g·kg^-1)daily for 5 days.One hour after the last injection,peripheral blood was harvested under steriled condition from rat left ventricular under general anesthesia.Mononuclear cells were isolated by density gradient centrifugation with Ficoll from peripheral blood,and cultured in?-MEM medium containing 10%FBS,2 mmol/L glutamines and 0.01 g/mL basic fibroblast growth factor?bFGF?in 25 cm² disposable plastic culture flask.The?-MEM medium volume was 3 mL.Culture condition was 37?,5%CO₂ and saturated humidity.The cells were subcultured when growth convergence degree exceeded more than 80%to conduct a purified culture of MSCs to the P3generation.The P3 generation cells were identified by flow cytometry?CD90,CD29,CD45,CD11b and CD79a?and immunocytochemical staining?the phenotype markers were CD34,CD73,CD105 and HLA-DR?.2.The isolation,culture and identification of BMMSCs in rats:After the rats were mobilized as descriped before,both femurs of each animal,were havesetd under sterile condition.The bone marrow cavity was washed by?-MEM medium containing 2%FBS with help of a syringe to obtain the single-cell suspension.After centrifugation,the supernatant was discarded,the cells were cultured with?-MEM and subcultured to the P3 generation.The BMMSCs phenotypic identification method was the same as PBMSCs part.3.Isolation and culture of M0macrophages:?1?Preparation of L929 medium?L929-CM?:L929 cells?about 1×10⁵/mL?were cultured in HG-DMEM medium containing 10%FBS for 7days.The supernatant was collected,filted with 0.25?m filter and stored in-80°C.?2?Isolation and culture of the M0macrophages derived from rat bone marrow:Femurs were collected under sterile conditions after SD rats were sacrificed.The bone marrow cavity was washed by RPMI-1640 medium.After centrifugation,the supernatant was discarded,macrophages medium?including 10%FBS,20%L929 conditioned medium RPMI-1640?was applied and cell density was adjusted to 2×10⁶/mL.Macrophages were seeded in 6 wells plate and cultured for 6 days to gain M0,the bone marrow-derived macrophages.4.The co-culture of MSCs and M0 macrophages:Purified bone marrow-derived macrophages were inoculated in 0.4?m Transwell culture plate with a density of 1×10⁵/cm².Then it was divided into 5 groups:PBMSCs group,BMMSCs group,macrophage?M0?group,PBMSCs and macrophage co-cultured group?PBMSCs+M0?,BMMSCs and macrophage co-cultured group?BMMSCs+M0?.PBMSCs/BMMSCs?1×10⁵/cm²?in the co-cultured group were inoculated in Transwell chamber,respectively.They were co-cultured with M0macrophages?1×10⁵/cm²?for 3 days.At the fourth day,the medium was changed into the serum-free medium.Supernatant was collected after 24 hours and stored in-80?waiting for cytokine detection by immunomicrosphere.5.Detection of co-cultured supe rnatant cytokines by immunomicrosphere:the collected supernatant was sent to Bio-Plex company and tested according to the Bio-Plex immune microsphere test kit manual.The second part:Regulation of the Th17/Treg cells balance via PBMSCs in vitro.1.Isolation and culture of rat spleen lymphocytes:spleen of SD rats?200-230g?was grinded gently with 5mL RPMI-1640 medium.The liquid was filted with 300 mesh nylon net.Suspensionwas collected after filtration.After Ficoll lymphocyte separation liquid was added,lymphocyte layer was collected after centrifugation.RPMI-1640 medium contained10%FBS,2 mmol/L glutamines.Lymphocytes were mixed with RPMI-1640 medium in25 cm² disposable plastic culture bottle.The RPMI-1640 medium volume was 3 mL.Culture environment was 37?,5%CO₂ and saturated humidity condition.2.The experiment groups:The ratio of lymphocytes to PBMSCs was 10 to 1.The total number of lymphocytes and PBMSCs was10⁷ and 10⁶.There were 3 groups:co-culture 5 days group,co-culture 10 days group and lymphocytes group.3.Cytokine contents in lymphocytes and PBMSCs co-culture supernatants were analyzed:The supernatant of each group was collected.Then the contents of TGF-?1,IL-17 and TGF-?1 in the supernatants of each group were detected by ELISA.4.The balance between Th17 and Treg cells system was detected in lymphocytes after lymphocytes co-cultured with PBMSCs.The expression of IL-6,Foxp3,ROR?T,TGF-?,IL17,IL-10 mRN A of lymphocyte was detected by qPCR.5.The changes of Th17 and Treg cell after lymphocytes and PBMSCs co-cultured:the percentages of Treg and Th17 cell subsets were analyesd by flow cytometry.Results:The part one:1.PBMSCs and BMMSCs had similar phenotypic characteristics.They expressed CD90,CD29,CD73 and CD105.They did not express CD45,CD11b,CD79a,CD34 and HLA-DR.2.Compared with PBMSCs group,macrophages co-cultured with PBMSCs/BMMSCs showed significantly higher IL-10 and IL-1?concentration?P<0.05?,lower TNF-?concentration in the sup ernatant?P<0.05?.Concentration of IFN-?had no significant change found in co-cultured group?P>0.05?.All cytokines in BMMSCs group were differed from PBMSCs group?P<0.05?.The part two:1.Result of ELISA suggested:concentration of TGF-?1 and IL-17 in lymphocyte group and co-cultured group could be detected by ELISA.The contents of TGF-?1 increased in co-cultured group,while IL-17 decreased?P<0.05?.There is no significant difference between 5 days and 10days group?P>0.05?.2.Fluorescence quantitative PCR data showed that:compared with the lymphocyte group,after co-cultured 5 days and 10 days,the expression of IL-6decreased?P<0.05?.IL-6 was a related gene of lymphocytes differentiation.Expression of IL-17 decreased in Th17 cell?P<0.05?.After 5 days,expression of Foxp3 and TGF-?1increased in co-cultured group,compared with lymphocyte,which was related gene of Treg cell differentiation?P<0.05?,and led to IL-10 expression increase?P<0.05?.The expression of Foxp3,TGF-?1 and IL-10 in co-cultured 10 days was significantly higher than the results of 5 days?P<0.05?.3.Results of flow cytometry showed that,the proportion of CD4⁺T cellsafter co-cultured 5 days and 10 days was higher than lymphocytes group?P<0.05?and the proportion of Th17 cells?CD4⁺IL17⁺?was lower than lymphocytes group?P<0.05?,but the proportion of Treg or Th17 had no difference for 5days and 10 days?P>0.05?.Conclusion:1.PBMSCs/BMMSCs co-cultured with macrophages could stimulate the secretion of IL-10 and IL-1?and reduce the secretion of TNF-?.BMMSCs showed stronger modifying ability than PBMSCs.Compared with BMMSCs,PBMSCs had different functions in immune regulation.2.PBMSCs could regulate related genes expression of Th17/Treg cells and increase the proportion of CD4⁺CD25⁺Foxp3⁺Treg cells,while inhibit the proportion of CD4⁺IL17⁺Th17 cel s in lymphocytes.