| Objective:To explore the potential role of programmed death-ligand1(PD-L1) expressed by the Kupffer cells in alleviating the liver transplantation acute rejection in echinococcus multilocularis infected rats. Methods:(1)The animals were divided into three groups:group A was injected subcutaneously infected group; Group B was infected of E.m by laparotomy. group C, percutaneous puncture E.m inoculation of liver. BN rats were3-4weeks old (n=20), each inoculated with2000Festival/ml of the Em suspension0.2ml. Observed rate of infection of each group at3months after infection, mortality, ELISA detection expression of Thl cytokines interferon gamma(IFN-y) and Th2cytokines white interleukin-10(IL-10) in the plasma.(2)The animal model of OLT were established with Kamada "double takes method" and divided control group:Lewis to BN (E.m uninfected) and experimental group:Lewis to BN (E.m infected) with30rats in each group. Six rats were randomly chosen in each group to observe the survival days. Six rats were respectively sacrificed at post-operative dayl,3,5and7and the peripheral blood and liver tissues samples were collected in each group. The pathological liver rejection classification was microscopically observed; peripheral level of aspartate aminiotransferase (AST) and total bilirubin (TBIL) levels were detected by Automatic Biochemical Analyzer. Plasma levels of Interluekin-10(IL-10) and interferon-y were detected by ELISA. The expression of PD-L1in transplanted liver tissue was detected by Immunohistochemistry and the expression of PD-L1m RNA was detected by real-time PCR assay.(3)Separation transplanted iver KCs of the experimental group and control group after7days and numbered with KCsE.m+and KCsE.m-. Meanwhile, separated and cultured splenic T lymphocytes (TCs) of normal BN rat. Cultured alone or mixed and which were divided into four groups:KCs experimental group:KCsE.m+; KCs the control group:KCsE.m-; mixed culture experimental groups:KCsE.m+TCs; mixed culture in the control group:KCsE.m-+TCs. Co-culture the KCs and TCs for24h and detect the proliferation of TCs by MTT; apoptosis of TCs by cytometry, and expression level of IL-10and IFN-y in supernate by ELISA. Results:(1)Observing the subcutaneous successful E.m infected rat model, and the death number was0, mortality was0%(0/20), the infection rate of60%(12/20), ELISA detection of three methods of three groups and the results show that peripheral serum of IFN-y, IL-10was no difference (P<0.05);(2)the steady and successful model of orthotropic liver transplantation was established in rats by using the modified Kamada method. The longest survival time in experimental group was26days with the median survival time was10.2±2.13; However by showing statistical significance, the longest survival time in control group was12days with the median survival time was22.5±4.04days. The post-operative levels of AST and TBIL in control group showed progressive increases and only a slight increase in experimental group. At the post-operative day7, the plasma level of IFN-y was significantly lower and the IL-10level was significantly higher in experimental group than that were in control group. PD-L1was highly expresses with wide extent in experimental group, however in control group, it was weakly expressed. In line with this, the PD-L1m RNA was higly expressed with wider extent in experimental group than that was in control group and showed statistical significance.(3)The PD-L1expression of group KCsE.m+was higher than KCsE.m-(P<0.05), the TCs proliferation in KCsE.m++TCs was (13258.34±951.26cpm), and lower than group KCsE.m-+TCs, which was (3047.98±101.42cpm), P<0.05. However, the apoptosis rate of group KCsE.m++TCs (8.83±0.37%)was significantly higher than that was in group KCsE.m-+TCs(1.34±0.16%), P<0.05. The expression of the level of the IFN-y in group KCsE.m++TCs of co-culture fluid was significantly lower than group KCsE.m-+TCs, P<0.05. However, the former expression (108.8±12.89pg/ml) of IL-10level was significantly higher than group KCsE.m-+TCs(49.13±17.63pg/ml), P<0.05. Conclusion: Echinococcus multilocularis infection may alleviate the post-liver transplantation acute rejection by up-regulating the level of PD-L1in KCs and inhibit the proliferation and accelerate the apoptosis of TCs. |
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