| Objection To separate the Wistar rat's bone marrow derived mesenchyal stem cell(BM-MSCs),and observe the effect that BM-MSCs modulate the balance of,provide the theoretical basis for the future treatment of complication caused by organ transplantation.Methods Choose SPF male Wistar rat for separating the MSCs and spleen mononuclear cell(SMNC).Culture and generate the BM-MSCs,then identify cell surface antigen by the Flow Cytomety.By morphology,adipogenic and osteogenic differentiation to judge whether the BM-MSCs is satisfied to the experiment.Separate the SMNC by the Magnet Activated Cell Sorting(MACS)to get CD4+T cells that are satisfied for the experiment.Co-culture the CD4+T cells with BM-MSCs to explore the ability that BM-MSCs modulate the balance of.Explore particular cytokines' affect on the BM-MSCs' ability of modulating the balance of by adding cytokine blocker.Add immunosuppressive drug to explore its impact on BM-MSCs modulating the balance of.All CD4+T cells will be analysised by the Flow Cytometry after staining the surface antigen.Supernatant will be tested by the Cytometric Bead Array(CBA)to analysis the cytokines' concentration.Result(1)Separation and culture of MSCs to the 4th generation,cell morphology called star or spindle,osteogenic differentiation VonKossa staining visible black calcium nodules,into fat differentiation oil red O staining can be seen intracellular orange fat droplets,flow The positive rate of CD90 and CD29 was> 95% and the positive rate of CD45 was less than 5%.The results showed that the obtained cells were relatively pure MSCs.The spleen mononuclear cells isolated by magnetic beads showed that CD4 positive rate was>95%,and it was proved that CD4+T cells were satisfied after the separation by magnetic beads.(2)MSCs cultured with different concentrations of MSCs and CD4+T cells for 72 hours had a short-term effect on the Th17 cells at 0.5 times the basal concentration,and 4 times the basal concentration of MSCs had a significant inhibitory effect on Th17 There was no statistically significant difference in cytokine IL-17.In addition,the proportion of Treg was not statistically different,but the level of IL-10 increased with the concentration of MSCs increased to 2 times the basal concentration reached the highest level,in addition,IL-6 and TGF-? There was no statistical difference in the level.(3)There was no significant difference in the proportion of Th17 between the TGF-? blockers,but the IL-17 was significantly increased in the high concentration group.The ratio of Treg was lower than that of the control group,but the Treg.The levels of IL-10 and IL-6 in the high concentration of TGF-? blocker were higher than those in the low concentration and the control group.The TGF-? group was not significantly different Statistical differences.After the application of high concentrations of IL-2 blockers,the proportion of Th17 was significantly lower than that of the low concentration group and the control group,and no effective concentration of IL-17 was detected in the culture medium to which the blocking agent was added.The levels of IL-10,IL-6 and TGF-? were not significantly different in the control group compared with the control group.(4)When FK506 was applied,the proportion of Th17 was not significantly different from that of the control group,and the proportion of Treg was lower than that of the control group.The levels of IL-10 and IL-6 in the culture medium were lower than those in the control group,but the concentration of IL-17 and TGF-? was not statistically different from the concentration of FK506.The proportion of Th17 cells was lower than that of the control group,and the proportion of Treg was higher than that of the control group,but there was no significant difference between different concentrations of rapamycin.IL-10 and IL-6 were significantly lower than those in the control group,but there was no gap between the different concentrations of rapamycin and TGF-? There was no statistical difference.Conclusion(1)BM-MSCs' modulation ability on Th17 has a slight effect with its concentration,and has limited effect on the secretion of IL-17 and IL-6.The positive regulation of Treg and its related cytokines TGF-beta was not obvious,but it could promote the secretion of IL-10.(2)blockage of TGF-? receptor can reduce the effect of BM-MSCs on the differentiation of Treg,but has no effect on the proportion of Th17.Also,the expression of IL-17,IL-10 and IL-6 was increased,which indicated that TGF-? could inhibit the expression and secretion of these cytokines.Block IL-2 receptor could effectively inhibit the proliferation and differentiation of Treg,meanwhile the proportion of Th17 was decreased.The expression of IL-17 could be strongly inhibited.In addition,there was no statistical difference between each groups of IL-10,IL-6 and TGF-?.The expression of these cytokines may not be regulated by IL-2.(3)in the presence of tacrolimus,the inhibition of BM-MSCs on Th17 was not affected,at the same time,the expression of IL-17 cytokines related to Th17 compared with the control group had no difference;but the ratio of Treg decreased compared with the control group,indicating a completely inhibitory effect of tacrolimus on Th17 and Treg.The expression of IL-17 and IL-6 in the supernatant was inhibited,which indicated that tacrolimus could inhibit the expression of inflammatory cytokines,and the increase of IL-10 indicated that tacrolimus could enhance the effect of BM-MSCs on the secretion of IL-10.After rapamycin treatment,the proliferation of Th17 was effectively inhibited and the expression of IL-17 was strongly inhibited.In addition,rapamycin can enhance the ability of BM-MSCs to promote the differentiation of Treg,but it can inhibit the secretion of IL-10. |
PDF Full Text Request