| Objective By studying the γδT lymphocyte immune response in peripheral blood mononuclear cells of chronic echinococcosis patients and secondary infection BALB/c mici to explore the mechanism of γδT lymphocyte immune function in chronic echinococcosis patients, in order to provide new evidence for further investigate the immune system of anti-hydatid disease and prevention and cure of hydatid disease.Methods(A)Detect the role of γδT cells in chronic echinococcosis patients: Detect the γδ T cells from peripheral blood mononuclear cells(PBMCs) of chronic echinococcosis patients by FACS. Isolate γδ T cells from peripheral blood of chronic CE patients using FITC-γδ TCR antibody for sorting by FACS, then expand theγδ T cells in condition with IL-2/anti-TCRγδ in vitro. Purified the r Eg14-3-3 then identified by western blot. Detect the response of γδ T cell to varying components of hydatids isolated from patients and recombinant E.granulosus antigen 14-3-3 by MTT assay and FACS. Detect several cytokines(included IFN-γ, TNF-α, IL17 A, TGF-β and IL-2) using culture supernatant of γδ T cells via stimulation with varying E.granulosus antigens by ELISA. Detect the m RNA expressions of bioactive molecules(IFN-γ/Perforin/Granzyme A and B/Fas L/TNF-α m RNA) in expanded γδ T-cells from 21 chronic CE patients in situation of stimulation of protoscoleces antigens by real-time RT-PCR.(B) Detect the change of γδT cells in secondary infection BALB/c mici: Collect cysts of Echinococcus granulosus establish secondary infection BALB/c mici and establish control group.Detect the model mice in feeder 8 months later.Extract the IEL by Percoll density centrifugation in model mice and detect the IEL and γδT cells by Flow cytometry.Statistics and analysis the change between infected group and control group on numbers of γδT cells and IEL.Results The results of γδT cells in chronic echinococcosis patients: Peripheral blood γδ T cells significantly reduced in the chronic CE(p<0.01), especially the Vδ2 subset as the main type of γδ T in peripheral blood(p<0.01). The expansion of γδ T cells was significantly activated in condition with IL-2/anti-TCRγδ in vitro. The proliferation of γδ T cells was apparently increased by antigens derived from protoscoleces components. IFN-γ concentration were obviously increased in supernatant of proliferated γδ T cultured for 3 days in comparison with the Ig G isotype control(P<0.01). Meanwhile, there’s no statistical difference of IL-17 A, TGF-β, IL-4, TNF-α, and IL-2 secretion was observed between cells treated with and without protoscoleces(P>0.05). The quantitative RT-PCR revealed that levels of these cytotoxic molecules(perforin, granzyme-A and B, IFN-γ and Fas L) expressed increasingly when interaction with protoscoleces(P<0.01)The results of γδT cells in secondary infection BALB/c mici: Echiococcus granulosus mice model established successfully, IEL+ γδT was reduced significantly, the number of IEL also reduced significantly.Conclusion In vitro, protoscoleces antigens can effectively induce the γδT cell proliferation in peripheral blood mononuclear cells(PBMC) of chronic echinococcosis patients. The m RNA expression and secretion of the main cytokine of IFN-γ was significantly higher and the m RNA expression of perforin, Fas L, granzyme A and B were significantly higher. Illustrate the γδT cells participate in the host immune response, and have anti-inflammatory effects in Echinococcus granulosus infection. In vivo, the γδT cells were significantly lower in PBMC of chronic echinococcosis patients, especially Vδ2+γδT cells. The s IEL+ γδT cell and the mall intestine IEL decreased significantly compared to the control group at the stage of chronic infection in secondary infection BALB/c mici. Illustrate that the γδT cells are inhibited at chronic infection stage. |
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