| BackgroundAt present,the related complications of acute rejection(AcR)are still one of the most important obstacles affecting the long-term survival of the receptors.Timely clearance of apoptotic cells is closely related to the formation of immune tolerance microenvironment.Kupffer cells(KCs)are professional phagocytes in the liver,and are also the main phagocytes to remove apoptotic cells.Previous studies have confirmed that KCs-mediated apoptotic cell clearance is an important factor in maintaining liver immune tolerance,but the molecular mechanism of how KCs mediates timely and effective apoptotic cell clearance and induces immune tolerance is still unclear.Recognition of phosphatidylserine(PtdSer)receptors on the surface of phagocytes is a key step in the phagocytosis of apoptotic cells Recently,it has been found that one of the PtdSer receptors,scavenger receptor family class F member 1(SCARF 1),plays a key role in regulating the clearance of apoptotic cells,and its regulatory effect is stronger than that of the previously discovered PtdSer receptors.However,relevant researches mainly focus on autoimmune diseases and have not yet involved in the field of transplantation.Therefore,to explore the role of KCs-mediated phagocytic clearance of apoptotic cells in inducing immune tolerance of liver transplantation and its specific mechanism can provide a new strategy for the prevention and treatment of AcR after liver transplantation and the induction of immune tolerancePart ? The effect of SCARF1 expression changes on the regulation of KCs immune functionObjective:to observe the effect of changes in SCARF 1 expression on the phagocytic function and cell phenotype of KCs and the mechanism by inhibiting or up-regulating the expression of SCARF 1 in KCs.Methods:(1)KCs were isolated and cultured according to the steps of type VI collagenase digestion,gradient centrifugation and selective adherent,and its phagocytosis was identified by ink swallowing experiment.(2)the successfully isolated and identified KCs were divided into four groups:the AdV-SCARF1 group;Ctrl-AdV group;shRNA-SCARF 1 group;Ctrl-shRNA group.Each group of KCs was transfected with AdV-SCARF1,shRNA-SCARF 1,Ctrl-AdV,Ctrl-shRNA lentivirus respectively.(3)immunofluorescence was used to test the efficiency of virus transfection;(4)the expression and transcription of SCRAF1 in the transfected KCs were detected by Western blot and RT-PCR.(5)the early apoptotic T cells were prepared by UV irradiation and co-cultured with the transfected KCs.(6)the number of KCs phagocytic apoptotic T cells in each group after co-culture was detected by immunofluorescence staining.(7)flow cytometry was used to detect the number of remaining early apoptotic T cells in the supernatant.(8)flow cytometry was used to detect the number of M2 cells and the expression of costimulatory molecule CD86 in each group of KCs.(9)the expression and transcription levels of IL-1?,IL-6,TNF-?,IL-10 and TGF-? in the supernatant of KCs in each group were detected by ELISA and RT-PCR.(10)the expression levels of IL-10,JAK,STAT3 and phosphorylated protein in KCs of each group were detected by western blot and immunofluorescence.Results:(1)KCs had normal morphology,good growth condition and good adhesion.(2)Immunofluorescence showed that the green fluorescence reached the peak in KCs after 48 h of transfection.(3)the results of western blot and RT-RCR showed that the expression of SCARF 1 protein in the AdV-SCARF1 group was significantly higher than that in the Ctrl-AdV group,while the expression of SCARF 1 protein in the shRNA-SCARF1 group was significantly lower than that in the shRNA-Ctrl group.The results of RT-PCR showed the same trend.(4)immunofluorescence results showed that the number of PI-positive KCs in the AdV-SCARF1 group was significantly higher than that in the Ctrl-AdV group,and the number of PI-positive KCs in the shRNA-SCARF1 group was significantly lower than that in the shRNA-Ctrl group.Flow cytotometry showed that the number of remaining apoptotic T cells in the AdV-SCARF1 group was significantly lower than that in the Ctrl-AdV group,and the number of remaining apoptotic T cells in the shRNA-SCARF1 group was significantly higher than that in the shRNA-Ctrl group.(5)Flow cytometry results showed The M2 KCs in AdV-SCRAF1 group were significantly higher than the Ctrl-AdV group.The proportion of M2 KCs in shRNA-SCARF1 group was significantly lower than that in shRNA-Ctrl group.The costimulatory molecule CD86 was significantly lower in the AdV-SCRAF1 group than that in Ctrl-AdV group.(6)ELISA and RT-PCR showed that the expression and transcription levels of f IL-1?,IL-6,TNF-? in the AdV-SCARF 1 group were significantly lower than those in the Ctrl-AdV group,while the expression and transcription levels of IL-1?,IL-6,TNF-? in the shRNA-SCARF1 group were significantly higher than those in the shRNA-Ctrl group.(7)Western blot results showed that the expressions of SCARF 1,p-JAK and p-ATAT3 in the AdV-SCARF1 group were significantly higher than those in the Ctrl-AdV group,and the expressions of SCARF 1,p-JAK and p-STAT3 in the shRNA-SCATF1 group were significantly lower than those in the shRNA-ctrl group.Conclusion:(1)overexpression of SCARF 1 in KCs can significantly increase the phagocytosis of apoptotic T cells by KCs.(2)Up-regulation of SCARF 1 promoted the activation of JAK/STAT3 signaling pathway which may leaded to M2-type polarization.(3)Up-regulation of SCARF 1 increased the secretion of anti-inflammatory factors and decreased the secretion of pro-inflammatory factorsPart ? Effects of SCARF1 on AcR and in the induction of immune tolerance after liver transplantation in rat modelsObjective:to investigate the role of SCARF 1 expression in KCs in immune tolerance after liver transplantation after transfection with adv-scarfl or scarf1-shrna by portal vein infusion.Methods:(1)acute rejection model of Lewis?BN rat liver transplantation was established by modified "double-sleeve method".(2)the receptors were randomly divided into three groups(n=20):AdV-SCARF1group,shRNA-SCRAF1 group,Control group.(3)10 rats in each group were randomly selected to observe the average survival time and survival rate of each group,and liver and blood samples of other 10 rats were collected for follow-up experiments.(4)the serum ALT,AST and TBIL levels of rats in each group were detected by automatic biochemical analyzer,and the liver function of rats in each group was evaluated.(5)histopathology was used to detect the degree of pathological injury of liver tissue in each group of rat;(6)TUNEL assay was used to detect the clearance degree of apoptotic cells in liver tissues of rats in each group.(7)the number of M2-type KCs in liver tissues of rats in each group was determined by immunohistochemistry.(8)the expression levels of IL-1?,IL-6,TNF-?,IL-10 and TGF-? in the serum of rats in each group were detected by ELISA.(9)expression levels of JAK,STAT3 and phosphorylated protein in liver tissues of rats in each group were detected by western blot.Results:(1)the mean survival time and survival rate of the receptor rats in the AdV-SCARF1 group were significantly improved compared with the other two groups.(2)serum AST,ALT and TBIL levels in the AdV-SCARF1 group were significantly lower than those in the other two groups.(3)histopathological examination results showed that acute rejection degree of liver tissues in the rats with the AdV-SCARF1 receptor group was significantly lower than that of the other two groups,while RAI score of liver tissues in the rats with the adv-scarfl receptor group was also significantly lower than that of the other two groups.(4)TUNEL results suggested that the number of apoptotic cells in liver tissues of the AdV-SCARF1 group was significantly lower than that of the other two groups.(5)immunohistochemical results suggested that the number of M2-type KCs in liver tissues of the adv-scarfl group was significantly higher than that of the other two groups.(6)ELISA results indicated that the expression levels of IL-1B,IL-6,TNF-a in serum of the receptor rats in the AdV-SCARF1 group were significantly lower than those in the other two groups,while the expression levels of IL-10 and TGF-B were significantly higher than those in the other two groups.(7)the results of Western blot indicated that the expressions of SCARF 1,p-JAK and p-STAT3 in liver tissues of the AdV-SCARF1 group were significantly higher than those of the other two groups.Conclusion:(1)overexpression of SCARF 1 in KCs significantly extended the mean survival time of acute liver transplantation rejection model in rats,and improved the survival rate of acute liver transplantation rejection model in rats.(2)overexpression of SCARF 1 in KCs significantly reduced liver tissue pathological damage and liver function in acute rejection model of liver transplantation in rats;(3)overexpression of SCARF 1 in KCs significantly increased the clearance of apoptotic cells by KCs in transplanted liver tissues;(4)overexpression of SCARF 1 in KCs significantly increased the number of m2-type KCs in transplanted liver tissue,decreased the expression of pro-inflammatory factors such as IL-1,IL-6,TNF-?,and increased the expression of anti-inflammatory factors such as IL-10 and TNF-? in serum.(5)The main mechanism of SCARF 1-induced immune tolerance in liver transplantation in transplanted liver KCs may be the activation of JAK/STAT3 signaling pathway. |
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