| Objective: Alveolar echinococcosis(AE)is a malignant and fatal parasitic disease caused by the larvae of Echinococcus multilocularis(E.m),which is known as "parasitic cancer".A large number of studies have shown that the infection of Echinococcus multilocularis(E.m)can cause the body to be in the state of immune tolerance and thus evade the attack of the host’s immune system.mediating the abnormal function of NK cells in liver of AE patients.Methods: Part one,The pathological manifestations of the proximal and distal hepatic lesions in AE patients were observed by pathological methods from 27 liver surgery specimens of AE patients.The degree of hepatic fibrosis was detected by Masson staining.The expression of molecules of KIR2DL1 in the proximal and distal hepatic lesions in AE patients was detected by immunohistochemistry.The expression levels of KIR2DL1 molecules at the proximal and distal ends of lesions in the liver of 17 AE patients were detected by flow cytometry,and the expression levels of relevant functional cytokines in NK cells were also detected.In the second part,a mouse model of E.m infection by hepatic portal vein was established.the size and number of liver surface lesions were observed and analyzed at different time periods(4 weeks,12 weeks,24 weeks).The changes in The expression of Ly49 A on the surface of NK cells in the liver region were detected by flow cytometry in mice infected with E.m at different time periods.Part three: Liver injury changes in AE patients were detected by controls.Phenotype and function of NK cells in AE patients were detected by flow cytometry and(PCR).Echinococcus multilocularis cyst fluid(EMF)was used to intervene the progress of human monocytic leukaemia cell THP-1 differentiation.In a co-culture system with NK cells and EMF-THP-1 cells,we analyzed the function of NK cells by enzyme-linked immunosorbent assay(ELISA)with or without antibody against KIR2DL1.Results: a large amount of lymphocyte infiltration.Masson staining results indicated that the degree of fibrosis was significantly higher near the liver lesion than far(Close vs Distance: 11.75±0.99%vs 2.25±0.27%,P < 0.0001).Immunohistochemical results showed that KIR2DL1 expression was significantly higher in the proximal liver tissues than in the distal liver tissues(Close vs Distance: 19.37±1.09%vs 5.63±0.39%,P<0.0001).Flow cytometry results showed that KIR2DL1 expression was significantly higher in CD56+NK cells than in the distal liver tissues of AE patients(Close vs Distance: 8.49±0.91%vs 5.17±0.40%,P<0.05).The proportion of Granzyme B secreted by the proximal liver of KIR2DL1+NK cells is significantly lower than that of the distal liver(Close vs Distance: 31.20±4.01%vs 50.69±3.16%,P<0.05).The secretion of effector molecule IFN-γ in the adjacent liver of KIR2DL1+NK cells was significantly lower than that in the distal liver(Close vs Distance: 4.71±0.79%vs 9.80±1.74%,P<0.05).In the second part,A mouse model of E.m transhepatic portal venous infection was successfully established.The results of Sirius red staining indicated that the degree of liver fibrosis in the metacercaria infection group was significantly higher than that in the sham group at 4 weeks after infection(P<0.001).At 12 weeks of infection,liver fibrosis was significantly higher in the metacercaria group than in the sham group and at 4 weeks of infection(P<0.001 or P<0.01).At 24 weeks of infection,liver fibrosis was significantly higher in the metacercaria-infected group than in the sham group and at 4 weeks of infection(P<0.001).ALT and AST levels in peripheral blood of the infected mice were detected at different time periods.The results suggested that ALT levels in peripheral blood of the infected mice were significantly higher than those of the sham group at 12 weeks after infection(control vs EM: 25.14±1.36 vs 37.26±2.85U/L,P<0.01).At 24 weeks after infection,the ALT level in peripheral blood of the infected group was significantly higher than that of the sham group(control vs EM: 26.16±1.47 vs 49.66±0.92U/L,P<0.01).At 24 weeks after infection,ALT levels in peripheral blood of mice were significantly higher than those at 4 and 12 weeks(4 weeks vs 12 weeks vs 24 weeks: 32.72±4.36 vs 37.26± 2.85 vs 49.66±0.92 U/L,P<0.05 or P<0.01).At 24 weeks of infection,the level of AST in peripheral blood of the infected group was significantly higher than that of the sham group(control vs EM: 114.70±1.59 vs 141.70±3.55U/L,P<0.01).At 24 weeks of infection,peripheral blood AST levels were significantly higher than at 4 and 12 weeks(4 weeks vs 12 weeks vs 24 weeks: 112.50±3.91 vs 118.50±2.75 vs 141.70±3.55 U/L,P<0.05 or P<0.01).Flow cytometry was used to detect the expression of Ly49 A molecules and related cytokines on the surface of NK lymphocytes in the liver of mouse models infected with metacercaria at different time periods.In the middle and late stage of infection(12 weeks and 24 weeks),Ly49 A expression was significantly higher than that in the sham group(P<0.05).Secretory effector cytokines Granzyme B,IFN-γ and TNF-a were significantly reduced at 12 and 24 weeks of infection(P<0.01 or P<0.05).Part three,Changes of liver injury in AE patients were detected,and the results showed that ALT and AST levels in peripheral blood of AE patients were significantly higher than those in the normal control group(HL vs AE: ALT,23.71±5.78 vs 59.4±13.45U/L,P<0.05).AST,18.33±2.00 vs 37.49±7.3U/L,P<0.05);The proportion of CD56 dimNK cells in the blood of AE patients was decreased by flow cytometry(P<0.05).Real-time quantitative PCR results suggested that the secretion of IFN-γ,Granzyme B and CD69 in NK cells of AE patients was significantly reduced compared with that of healthy controls,but the expression of KIR2DL1 was abnormally increased.Argininase-1,CD206 and CD163,the markers of macrophage M2 polarization in the treatment group,were all higher than those in the healthy control group(P<0.001).After incubation of these emf-Thp-1 cells with NK cells isolated from AE patients,the function of NK cells was significantly inhibited,thus causing cytotoxicity.However,this effect was basically eliminated after the introduction of anti-KIR2DL1 antibody.The results showed that the infection of metacercaria can regulate macrophages through KIR2DL1 and induce NK cell dysfunction.Conclusion: 1.In the liver regional immunity of AE patients,the surface inhibitory receptor KIR of the NK cells is significantly overexpressed,which is transmitted to the inhibitory signal transmission,resulting in the reduced function of local NK cells in the liver.2.E.m infection can regulate macrophages through KIR2DL1,induce NK cell dysfunction,lead to the depletion of NK cell function,and cause chronic parasitism and long-term coexistence of E.m infection in the liver of host. |
PDF Full Text Request