| Objective: To establish the model of abdominal cardiac allograft transplantation in rats, andinject bone marrow mesenchymal stem cells of donor and IL-6monoclonalantibody(IL-6mAb) via jugular vein.To study how MSCs and IL-6mAb affect theCD4~+CD25~+foxp3~+T cells’ proportion, Foxp3protein’s expression rate, Th17cells’ rate andthe donor heart survival time to receptors. Besides, combined with the experiment in vitrowhich MSCs and CD4~+T cell co-cultured under different conditions, to reveal themechanism of MSCs and IL-6monoclonal antibody on immune tolerance.Methods: The external part: MSCs were cocultured with allogenenic CD4~+Tlymphocytes in different conditions and different proportions, the groups of the experiment:Group A (CD4~+T cultured alone), Group B1(MSCs/CD4~+T1:1), Group B2(MSCs/CD4~+T1:10),Group B3(MSCs/CD4~+T1:100), Group C (MSCs/CD4~+T/IL-6mAb1:10), GroupD (MSCs/CD4~+T/TGF-βmAb1:10),Group Bt Transwell (MSCs/CD4~+T1:10), Group CtTranswell(MSCs/CD4~+T/IL-6mAb1:10), Group Dt Transwell(MSCs/CD4~+T/TGF-βmAb1:10).To observe cell morphology with an inverted microscope, detect the ratio ofCD4~+CD25~+regulatory T cells (Treg), the proportion of Foxp3protein expression andchanges in the proportion of Th17cells with flow cytometry.To detect TGF-beta1, IL-10,IL-2, IL-6, IL-17in the supernate with ELISA.Internal parts: Donor(SD rats) and receptor(Wistar rats) of20were randomly dividedinto four groups, GroupⅠ(control group): The heart from SD rat was transplanted into theabdomen of Wistar rat and receptor without any treatment. GroupⅡ (PBS group):receptors of Wistar rats were injected PBS daily0.2ml before surgery for five consecutivedays and after heart transplantation for3days via jugular vein. Group III (MSCs group): receptor was injected mesenchymal stem cells (MSCs)1×10~6via the neck vein beforepreoperative5th day and after surgery for3days.Group Ⅳ (MSCs and anti-IL-6mAbgroup): receptor Wistar rat were injected MSCs before heart transplantation for5days andwere injected IL-6mAb for five days and injected MSCs for three days after thetransplantation by jugular vein (IL-6mAb100μg/kg/day). Testing Items: cardiac allograftsurvival time, donor heart pathological changes, receptor of CD4~+CD25~+foxp3~+Treg cellchange, Th17cells’ change,the expression of Foxp3protein in the Treg cells and serumTGF-β1,IL-10, IL-2, IL-6, IL-17change.Results: The external part:(1) the results of CD4~+CD25~+Treg,the expression ofFoxp3protein on the Treg cells and the proportion of Th17cells in Supernatant fluid: TheCD4~+CD25~+Treg of Group C、Ct in the Supernatant fluid were significantly higher than theother groups(P<0.05). The CD4~+CD25~+Treg of Group C was significantly higher than thegroup Ct(P<0.05);The Foxp3protein expression of group C and Ct were significantlyhigher than the other groups(P<0.05).The Foxp3protein expression of group C wassignificantly higher than the group Ct(P<0.05).The Th17cells of group C and Ct weresignificantly lower than the other groups(P<0.05);The Th17cells of group C wassignificantly lower than group Ct(P<0.05).(2) The results of receptor TGF-β1insupernatant fluid:The TGF-β1concentration in the supernatant of group C wassignificantly higher than other groups(P<0.05).(3) The results of receptor IL-10insupernatant fluid: The Group C and Group Ct in the supernatant of IL-10concentrationswere significantly higher than the other groups(P<0.05). The Group C was significantlyhigher than the group Ct(P<0.05).(4) The results of receptor IL-2in supernatant fluid: TheIL-2concentration in the supernatant of group C and group Ct were significantly lowerthan the other groups(P<0.05). The Group C was significantly lower than the groupCt(P<0.05).(5) The results of receptor IL-6in supernatant fluid:The IL-6concentration inthe supernatant of group C and group Ct were significantly lower than the othergroups(P<0.05).The group Ct was significantly lower than the groupC(P<0.05).(6) Theresults of IL-17in vitro experiments: The IL-17concentration in the supernatant of groupC and group Ct were significantly lower than the other groups(P<0.05).There were nosignificant differences between group C and groupCt.Internal parts:(1) The donor heart mean survival time (MST):The average survival time of group III and group IV were longer than the groupⅠand groupⅡ(P<0.05). Theaverage survival time of group Ⅳ was longer than the group Ⅲ(P<0.05).(2) The results ofreceptor CD4~+CD25~+Treg, the expression of Foxp3protein in the Treg cells and theproportion of Th17cells in peripheral blood:The peripheral blood CD4~+CD25~+Treg ofGroup Ⅲ and group Ⅳwere significantly higher than group Ⅰ and groupⅡ(P<0.05). Theperipheral blood CD4~+CD25~+Treg of Group IV was higher than group III(P<0.05). Theperipheral blood Foxp3protein expression rate of group Ⅳwere significantly higher thanthe other groups(P<0.05). There is no statistically significant difference between group I, II,III. The peripheral blood th17cell ratio of Group Ⅲ and group Ⅳwere significantly lowerthan group Ⅰ and groupⅡ(P<0.05). The group IV is lower than group III(P<0.05).(3)Theresults of TGF-beta1of receptor in peripheral blood:The concentration of Group III and IVwas significantly higher than group I and group II(P<0.05). There were significantdifferences between group III and group IV (P <0.05).(4) The results of IL-10of receptorin peripheral blood:The concentration of Group III and IV were significantly higher thangroup I and group II(P<0.05). The concentration of Group IV was significantly higher thanGroup III(P<0.05).(5) The results of IL-2of receptor in peripheral blood: Theconcentration of Group III and IV was significantly lower than group I and groupII(P<0.05). The concentration of Group IV was significantly lower than Group III(P<0.05).(6) The results of IL-6of receptor in peripheral blood:The concentration of Group III andIV were significantly lower than group I and group II(P<0.05).The concentration of GroupIV was significantly lower than Group III(P<0.05).(7) The results of IL-17of receptor inperipheral blood:The concentration of Group III and IV were significantly lower thangroup I and group II(P<0.05).The concentration of Group IV was significantly lower thanGroup III(P<0.05).Conclusion:1. When MSCs and IL-6monoclonal antibody were co-cultured with CD4~+T,the CD4~+CD25~+Foxp3~+Treg ratio would be improved significantly, and the proportion ofTh17cells would be reduced, survival time of heart can be extended. Meanwhile, itdose-related with the MSCs.2.Regulation mechanism of contact between MSCs and CD4~+T cells play an important role in immune tolerance.3. MSCs were mainly regulated by lower down the IL-2, IL-17and increase the expressionof IL-10,TGF-β1etc., so as to achieve the purpose of immune suppression. |
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