| Background and objective:Acute on chronic liver failure displayed sepsis-like immune paralysis,which was closely related with infection and multi-organ failure.The progression of clinical course had close relation with infection and infection was the common acute factors,leading to high motality.Our group had already successfully established an animal of Concanavalin A 5 times repeated stimulation with immune suppression and infectious complication.Myeloid-derived suppressor cells are a heterogeneous population of cells that expands during cancer,sepsis and infection,and that has a remarkable ability to suppress T-cell responses.These cells constitute a unique component of the immune system that regulates immune responses in the context of various diseases.However,in acute on chronic liver failure,the mechanism of myeloid-derived suppressor cells inducing the immune paralysis had not reported.And then we would explore the role of proinflammatory factor tumor necrosis factor–?played in the model of infection,in order to provide reference to the disease.Part One The mechanism of immune suppression in animal model of acute-on-chronic liver failure which is induced by Con AObjective:Our group had already successfully established an animal of Concanavalin A 5 times repeated stimulation.This model could simulate the immunosuppressive state of acute on liver failure.The patients tended to have a high infection and mortality with the immunosuppression.Our study tried to explore the mechanism of the immunosuppressive state in our animal model.The Specific steps were in the following:1)I had mastered Concanavalin A 5 times repeated injection and the animal model was established in skilled and high stability;2)to probe into the quantity and immunosuppressive function of myeloid derived suppressor cells;3)to investigate the mechanism of the immune paralysis induced by myeloid derived suppressor cells.Methods:1.We builded the model by retrobular injection Concanavalin A every 48 hours,a total of five times.The control group was given the same volume normal saline injection.The general status and survival of mice was observed.2.After 12 hours of each given the Concanavalin A administration,the mice were sacrificed and the viscera were taken.Detecting the absolute counts and the percentage of myeloid derived suppressor cells using flow cytometry instrument.3.After the injction for the last time,we took myeloid derived suppressor cells of the model,cocultured it with CD4~+T cells in vitro,and then detected the immunosuppressive ability of myeloid derived suppressor cells.Sorting out of the myeloid derived suppressor cells of the model and adopted it to the acute liver injury mice induced by once Concanavalin A administration.Through the testing of the liver function after 12 hours in order to identify the immunosuppressive function of myeloid derived suppressor cells indirectly.4.After 48 hours of the fifth administration of Concanavalin A,determining the expression of MHCII and PD-L1 on the myeloid derived suppressor cells.And then,detecting the expression of PD-1,CTLA-4,CD62L and apoptosis of CD4~+T cells.At last,the absolute counts and percentage of regulatory T cells were tested utilizing the flow cytometry instrument.Results:1.Myeloid derived suppressor cells of the model possessed the functions of immunosuppression in vivo and in vitro.2.Myeloid derived suppressor cells accumulated in the animal model,and in the spleen and bone marrow were mainly the granulocytes.3.The expression of MHCII on the myeloid derived suppressor cells reduced significantly and the expression of PD-L1 increased.4.CD4~+Tcells exhibited increased expression of of PD-1,CTLA-4,apoptosis and reduced expression of CD62L.And the regulatory T expanded in the liver.Conclusion:We researched the mechanism of immune paralysis induced by the myeloid derived suppressor cells in the animal model of acute on chronic liver failure.The state of immunosuppression were leaded by the accumulation of myeloid derived suppressor cells in the spleen and bone marrow and the expansion of regulatory T in the liver.The mechanism lied in myeloid derived suppressor cells expressing lower MHCII and higher PD-L1,inducing CD4~+T increased expression of PD-1,CTLA-4,apoptosis,reduced expression of CD62L,and the accumulation of regulatory T.Part Two The role of tumor necrosis factor –? played in animal model of acute-on-chronic liver failure complicated with secondary infectionObjective: Acute on chronic liver failure manifested the immune paralysis as sepsis,rsulting in suffering from infection.The progression of clinical course was related to secondary infection and infection was the common acute factors of the patients,both leading to high mortality.Our group had established the animal model of acute-on-chronic liver failure complicated with secondary infection.We aimed to further study the role of proinflammatory factor tumor necrosis factor –? played in this model.Methods: 1.We established the model by intraperitoneal injection of Escherichia Coli on the former model of acute on chronic liver failure.Control mice were given the same volume of normal saline.Survival rate and general situation were observed everyday.2.After injection of Escherichia Coli,we collected the blood at 3,12,24,48,72,96 hours,and checked the level of IL-6,IL-10,IL-12,TNF-?,IFN-?,MCP-1 by cytometry beads array.And after 24 hours of injection of Escherichia Coli,the liver were taken and made into paraffin slice.3.We administrated the mice of etanercept through abdominal cavity 16 hours ahead of the injection of Escherichia Coli,blocking the TNF-? in advance.The groups were in the following: 1)5 times of Concanavalin A with injection of both Escherichia Coli and etanercept;2)5 times of Concanavalin A with injection of Escherichia Coli;3)5 times of normal saline with injection of both Escherichia Coli and etanercept;4)5 times of normal saline with injection of Escherichia Coli.4.We sacrificed the mice and took the viscera,then measured the expression of TNF-?in the myeloid derived suppressor cells using the flow cytometry instrument.Results: 1.The minimum lethal dose of Escherichia Coli in animal model of acute-on-chronic liver failure complicated with secondary infection was confirmed.And the pathological damage of liver in the model was relatively slight.2.The proinflammatory and anti-inflammatory cytokine of the model were both higher than the control groups(normal saline with injection of Escherichia Coli),and the changes of proinflammatory factor TNF–? was particularly obvious.3.After neutralizing TNF-? with etanercept,the survival rate of the model group(5 times of Concanavalin A with injection of both Escherichia Coli and Etanercept)was significantly higher than the control ones.4.The expression of TNF-? was higher in the granulocytic myeloid derived suppressor cells(G-MDSC)than in the monocytic myeloid derived suppressor cells(M-MDSC).Conclusion: The animal model of acute-on-chronic liver failure complicated with secondary infection was in a more intense state of systemic inflammatory response and the minimum lethal dose of Escherichia Coli was confirmed.The TNF-? mainly came from granulocytic myeloid derived suppressor cells and played an important role in the immune injury. |
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