| Objective Construct a recombinant adenovirus which can overexpress TIPE2gene.Use the recombinant adenovirus pretreat the cardiac allograft model of heterotopictransplantation in rat so that it can overexprss TIPE2gene in the model, research the effectthat how the overexpressed TIPE2gene effect the recipients,s autoimmune regulator andallograft immune rejection. explore the possibility of inducing immune tolerance of therecipients in rat, and reveal its mechanism of action.Methods Construct The CDS region of rat,s TIPE2gene to the synthetic adenovirusvector AdC68, make it can overexpress TIPE2gene. Recipients SD rats and donor Wistarrats were randomly divided into five groups: Group A (blank group):The heart from Wistarrat was transplanted into the abdomen of SD rat without any pre-treatment; Group B(FK506group): After the heterotopic heart transplantation model in rat succeed, treat themodel rat with immunosuppressant FK5060.5mg/Kg.d continuously. Group C(recombinant group): After the heterotopic heart transplantation model in rat succeed, treatthe model rat with recombinant adenovirus1×1011VPs by tail vein injection continuously.Group D (FK506and recombinant adenovirus group): After the heterotopic hearttransplantation model in rat succeed, treat the model rat with half the amountimmunosuppressant FK5060.25mg/Kg.d and recombinant adenovirus1×1011VPs by tailvein injection continuously. Group E (non-associated virus group):After the heterotopicheart transplantation model in rat succeed, treat the model rat with non-associatedrecombinant adenovirus1×1011VPs by tail vein injection continuously. Observations: Themean survival time(MST) and pathological changes of the cardiac allograft；Separate theperipheral blood mononuclear cells (PBMC) of the recipient SD rats first seven days aftertransplantation at day7, The proportion of peripheral blood CD4+CD25+Treg and theexpression of TCR (T cell receptor) was detected by flow cytometry; the expression ofTNF-α、IFN-γ、IL-2、IL-4、IL-10in peripheral blood was detected by ELISA; The rate of TIPE2expression in the spleen and draining lymph nodes of the lower abdomen wasdetect by western blot; the expression of FoxP3mRNA in the draining lymph nodes of thelower abdomen was detected by Real-time PCR.Results The CDS region of TIPE2gene can construct to the Synthetic recombinantadenovirus vector AdC68successfully, and can overexpress TIPE2gene stably.(2) Themean survival time of donor heart (MST): group A:9±1.6d；group B：26.3±3.8d；group C：33.4±3.5d；group D：48±6.6d；group E：28.8±6.1d. Compare to group A, the MST of donorheart in the rest groups were prolonger significantly, P<0.05,all have statistical significance;group C compare with group B,the MST of donor was prolonger significantly,P<0.05,it hasstatistical significance;Compare with group A,B,C and E,the MST of donor heart in groupD was prolonger significantly,P<0.05,all have statistical significance; Group B comparewith E,P>0.05,it has no Statistical difference.(3) The results of receptor CD4+CD25+Tregin peripheral blood: Goup A:1.70±0.66%；group B：6.42±1.17%；group C：9.38±1.13%；group D：31.34±2.23；group E：1.70±0.72%；Compare with group A,B,C and E, theproportion of peripheral blood CD4+CD25+Treg in Group D was increased significantly,P<0.05, all have statistical significance; Group C compare with group B,the proportion wasincreased significantly,P<0.05,it has statistical difference;Compare to group A,theproportion was increased significantly in group B,C and D, P<0.05, all have statisticaldifference.(4) The expression of IL-2（pg/ml): group A:2007.32±464.67; groupB:1142.64±83.09；group C：735.59±194.55；group D：515.38±50.60；group E：915.76±180.60.The concentration of IL-2in group D were decreased significantly compare withgroup A,B and E, P<0.05, all have statistical significance;Group C compare with groupB,the The concentration of IL-2was decreased significantly,P<0.05,it has statisticaldifference;Compare to group A, the concentration of IL-2were decreased significantly inthe rest groups, P<0.05, all have statistical difference; Group B compare to groupC,P<0.05,it has statistical difference; Group D compare with group C, P>0.05,it has nostatistical significance.(5) The expression of IL-4（pg/ml): group A:27.26±21.29;groupB:136.53±55.54; group C:213.25±89.48; group D:444.37±36.73; group E:166.84±58.16.The concentration of IL-4in group D were increased significantly compare with group A,Band E, P<0.05, all have statistical significance;Group C compare with group B,the Theconcentration of IL-4was increased significantly,P<0.05,it has statistical difference; Compare to group A, the concentration of IL-4were increased significantly in the restgroups, P<0.05, all have statistical difference; Group B compare with group C and E,P>0.05,it has no statistical significance.(6) The expression of IL-10（pg/ml): groupA:13.92±4.17;group B:14.75±9.17;group C:32.70±10.01; group D:78.18±13.31;groupE:33.95±14.50.The concentration of IL-10in group D were increased significantlycompare with group A,B,C and E, P<0.05, all have statistical significance; Theconcentration of IL-10in group C were increased significantly compare with group B,P<0.05, it has statistical significance; Compare to group A, the concentration of IL-10wereincreased significantly in group C,D and E, P<0.05, all have statistical difference; Group Ccompare with group E and group A compare with group B, P>0.05,it has no statisticalsignificance.(7) The expression of TNF-α(pg/ml):group A：1665.43±209.06; groupB:821.97±271.40; group C:369.33±122.58; group D：214.80±35.84；group E：1148.99±140.63. In group D,the concentration of TNF-αwere decreased significantly compare withgroup A,B and E, P<0.05, all have statistical significance. the concentration of TNF-α wasdecreased significantly compare with group B, P<0.05, it has statistical significance.Compare to group A, the concentration of TNF-α were decreased significantly in the restgroups, P<0.05, all have statistical difference; Group D compare with group C, P>0.05,ithas no statistical significance.(8) The expression of IFN-γ(pg/ml): group A:6769.31±1659.95; group B:6290.93±1124.97; group C:3739.59±493.21; group D：2840.97±444.67;group E：5367.94±183.96.In group D,the concentration of IFN-γ were decreasedsignificantly compare with group A,B and E, P<0.05, all have statistical significance. theconcentration of IFN-γ in group C was decreased significantly compare with group B,P<0.05, it has statistical significance. Compare to group A, the concentration of IFN-γwere decreased significantly in the rest groups, P<0.05, all have statistical difference;Group D compare with group C, P>0.05,it has no statistical significance.(9) The result ofexpression level of FoxP3mRNA in the draining lymph nodes of the lower abdomen: theexpression level of FoxP3mRNA in group D was increased significantly compare with therest groups,P<0.05,it has statistical significance;but among the group A,B,C andE,P>0.05.there was no statistical difference.(10)The rate of TIPE2expression in the spleenand draining lymph nodes of the lower abdomen: the TIPE2expression in group C and Dwere increased significantly compare with the rest groups,but between the twogroups,there was no significant difference. Conclusions (1) TIPE2gene can be constructed to recombinant vector AdC68successfully, and the recombinant adenovirus can overexpress TIPE2gene stably in vivoand in vitro.(2) Use the recombinant adenovirus pretreat the cardiac allograft model ofheterotopic transplantation in rat so that it can overexprss TIPE2gene in the model, candown regulate the TNF-α、IFN-γ and IL-2express level, up regulate IL-4and IL-10express level, reduced the level of acute rejection, prolonger the donor heart,s survival timein the recipients.(3) The recipient SD rats which overexpressed TIPE2gege, Activated Tcell receptor signaling pathway in vivo, increased the propotion of CD4+CD25+Treg cells,reduced the level of acute rejection.(4) we observed in the heterotopic abdominal hearttransplantation model in rats: The effect for induce rat heart transplantation immunetolerance of recombinant adenovirus is superior to FK506, it can relieve the rejectionreaction more effective and prolonger the survival time of donor heart.(5) compare totreatment only with recombinant adenovirus, combined treatment with recombinantadenovirus and FK506can prolong the survival time of cardiac allograft significantly，bothcan better relieve acute vascular rejection (AVR) occurs, but there have no significantdifference between the two groups, both can not completely overcome the acute rejectionreactions. |
PDF Full Text Request