| BackgroundAlthough with the wide usage of immunosupressants, acute rejection isstill a major cause of early-stage failure of liver transplantation. So, inhibitionof acute rejection and inducement of immune tolerance are the best solutionsfor this puzzle. Recent studies showed the occurrence of acute rejection has notonly been relevent with imbalance of Th1/Th2, but also with Th17/Treg closely.Therefore, migration Th17to Treg may be another best way to induce immunetorence. Today, many reports regarding imbalance of th17/treg with kidney,heart, pancreatic islet, skin transplantation can be seen and only a few aboutliver transplantation, furthermore, the mechanism of Th17/Treg involvingacute rejection still remains obscure. So it is crucial to explore the effect andmechanism of Th17/Treg in transplant immunity and to find a new targetinducing tolerance for clinical liver transplantation.PartⅠEstablishment of acute rejection and torlerence models of livertransplantation in inbred ratsObjective: To establish stable models of liver transplant acute rejection andtorlerance in inbred rats, and explore the techniques of OLT and characteristics of those models. Methods: Orthotopic liver transplantations in inbred rats(LEW and BN) were divided into two groups:①LEW-BN (REJ);②BN-LEW(TOL)，and performed with modified kamada’s method.The recipients’ survivalrate was observed, and blood samples and hepatical tissue were obtainedrespectively at day1,3,5,7post transplantation. Liver function was measuredwith a biochemical analyser. The change of liver morphological was examinedby hematoxylin-eosin (HE) staining and reject activity index (RAI) wascalculated with Banff standard. Results:⑴Recipients in REJ group graduallybecame depressed, inactive, and anorectic and passed dark yellow urine. Butrecipients in TOL group were almost as active as normal. A Kaplan–Meiermodel was constructed from the data to compare overall survival rates betweenthe two groups. All animals in REJ group died within13days. The meansurvival time of TOL group (59.67±7.03days) was significantly longer thanthat of REJ group (11.32±0.83days)(P<0.05).⑵The liver function detectedat day1between two groups was no significant difference (P＞0.05), but serumlevels of ALT,AST and TBIL were higher obviously in REJ group from day3to day7than that of TOL group (P<0.05).⑶Serious morphological changeswere shown in hepatic tissue of REJ group compared with those of TOL groupfrom day3post-transplantation. Hepatocytes were severely degenerated andvacuolated; hepatic sinusoids were gravely expanded and filled with manyerythrocytes; portal areas were infiltrated with a few inflammatory cells;sinusoidal endothelial cells were seriously swollen. The RAI scores increased significantly compared with those of TOL group, consistent with the result ofmorphological changes (P<0.05). Conclusion: The model of REJ (from LEWtoBN) exhibits a typical acute rejection post-transplantation, and may beconsidered as an ideal one for study the acute rejection of liver transplantation,whereas TOL (from BN to LEW) may be a tolerance model.PartⅡThe changes of Th17/Treg related-cytokines in rats livertransplant acute rejection and toleranceObjective: To explore the imbalance of Th17/Treg related-cytokines in ratliver transplant AcR and tolerance. Methods: Inbred rats performed with OLTwere divided into two groups:①rejection group(REJ);②tolerance group(TOL). Blood samples and hepatical tissue were obtained at day1,3,5,7postoperatively. The proportion of Th17andTreg cells in peripheral bloodmononuclear cell (PBMC) were analyzed with FCM.Serum level of Th17related-cytokines (IL-17, IL-23and IL-6) and Treg related-cytokines (IL-10,TGFβ1) were assayed by ELISA. The protein levels of above-mentionedcytokines were examined by western blot, and RORγτ/FoxP3mRNAexpression were analyzed with RT-PCR. Results:⑴The proportion of Th17cells in PBMC was higher than that of Treg cells in REJ group (p<0.05),whereas there was no significant difference in TOL group.⑵RORγtexpression levels increased time-dependently and were much higher in the REJgroups than those in TOL groups at the time points (3,5and7day) post-transplantation (p<0.05). In contrast, Foxp3expression levels decreasedtime-dependently and were markedly lower in the REJ groups when comparedwith the TOL groups at the same points(p<0.05). But the RORγt and Foxp3expression levels were similar at day1post-transplantation in TOL and REJgroups.⑶The protein and mRNA levels of Th17-and Treg-related cytokinesin TOL groups were similar with those in REJ groups at day1post trans-plantation. However, the expression of Th17-related cytokines(IL-17, IL-23,IL-6) in REJ groups significantly increased when compared with day-matchedTOL groups and achieved a maximal value at day7after surgery(p<0.05), incontrast, the expression of Treg-related cytokines (IL-10, TGF-β1) wasmarkedly lower than those of TOL groups (p<0.05). Conclusion: Theimbalance of Th17/Treg between tolerance and rejection groups during ratliver transplantation, suggesting a potential role of Th17/Treg imbalance inpathogenesis of acute transplant rejection.PartⅢ The effect of adoptive transfer of CD4+CD25+Treg on ratliver transplant acute rejectionObjective: To study the effect of adoptive transfer of CD4+CD25+Treg cellson rat liver transplant acute rejection. Methods: CD4+CD25+Treg cells weresorted with MACS from SPMCs of BN, and stimulated by spleen lymphocytesof LEW. After demonstration of its efficiency of the sorted CD4+CD25+Tregcells, rejection recipients (BN) were treated with or without injection of CD4+CD25+Treg cells via tail vein, then divided into two groups: adoptivetransfer(AT) and non-adoptive transfer(NAT) group. Survival rate of recipientswas closely observed, and blood samples and hepatic tissue were obtained atday7postoperativly. Liver function was measured with an automaticbiochemical analyser and pathological changes of liver were analyzed by HEstaining.Serum levels of IL-17, IL-23, IL-10,TGF-β1,IL-4and IFN-γ wereassayed by ELISA. The protein levels of above-mentioned cytokines in hepatictissue were measured with western blotting. Results: Adoptive transfer ofCD4+CD25+Treg cells alleviated the degree of acute rejection, and thepostoperative survival rate in AT group was obviously higher than that of NATgroup (P <0.05). The results of Western Blot and ELISA exhibited that theexpression levels of IL-17,IL-23were remarkably lower in AT group than thoseof NAT group (P <0.05); In contrast, the expression levels of IL-10,TGFβ1weremuch higher in the AT group (P <0.05).Meanwhile, the expression of INF-γandIL-4were much lower in two groups, suggesting CD4+CD25+Treg maydirectly inhibit the proliferation of activated CD4+/CD8+T cells in such milieu.Conclusion: Adoptive transfer of CD4+CD25+Treg can alleviate acuterejection in rats mainly by up-regulating expression of typical Treg-relatedcytokines and down-regulation Th17-type cytokines expression, promotingTh17/Treg immune deviation formation, and partly by inhibition of Th1immunity simultaneously in favour of induction rat transplant tolerance. |
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