Study On The Inhibitory Effect Of Tim-4 To Murine Autoimmune Hepatitis

Partâ… STUDY ON THE INHIBITORY EFFECT OF Tim-4 TO MURINE AUTOIMMUNE HEPATITISThe TIM(T-cell immunoglobulin domain and mucin domain) gene family was first discovered in 2001 and received much attention due to its location on mouse chromosome 11B1.1,a genetic region associated with multiple diseases including asthma,allergy and autoimmune diseases.The Tim family consists of eight members in mice,encoding Tim-1 to Tim-4 and putative Tim-5 to Tim-8. Tim-1 is expressed on the surface of Th2 cells and activated T cells;Tim-2 is selectively expressed on the surface of Th2 cells and involved in the negative regulation of Th2 response in the proliferation and differentiation as well as the secretion of cytokines;Tim-3 is specifically expressed on the surface of Th1 cells,and its interaction with corresponding ligand serves to supress Th1 responses;Tim-4 proteins are only expressed on activated antigen-presenting cells(APCs),however,it is not fully understood for the regulatory role of Tim-4 to APCs.Recently,accumulating data suggest that Tims expressed on the surface of immunocytes are involved in regulating the function of immune system.Tim-4,unlike the other Tim molecules,is not expressed in T cells,but is expressed in APCs both at the mRNA and protein levels,particularly in mature myeloid-derived lymphoid dendritic cells and macrophages.The recent evidences showed that Tim-4 could inhibit or activate T cell-mediated immune response through binding to Tim-1 or other receptors.Furthermore,Tim-4 also acts as the receptor of phosphatidylserine and is involved in the clearance of apoptotic bodies,which suggests that Tim-4 expression on the surface of APCs may exert the effector function to these cells.To certify regulatory effects of Tim-4 to APCs,we have established the new macrophage line overexpressing Tim-4 molecules,and our previous studies in vitro indicated:macrophage line RAW264.7 expressed endogenous Tim-4 molecules,and Tim-4 mRNA and protein expression increased in a dose-dependent way after simulation with LPS;Tim-4 inhibited the activity of macrophage line RAW264.7 through down-regulating the secretion of NO,the expression of iNOS,TNF-αand the co-stimulatory molecules including CD80 and CD86.Based on the above experimental results,we aim to confirm Tim-4 may inhibit the activity of macrophage in vivo using autoimmune hepatitis model.Many investigations have shown that macrophages are very important in the murine acute hepatic injury model induced by ConA.So we further evaluate the roles of Tim-4 in vivo with this typical model.Firstly,endogenous macrophages were depleted by delivering GdCl₃.Then the macrophage line RAW264.7-Tim-4 and corresponding control cells were transferred to mice by i.v.Finally,mouse acute hepatic injury was induced by ConA.After sacrificing mice at different time point,we monitored hepatic injury by the secretion of serum ALT,HE staining, TUNEL and analyzed the potential mechanism through TNF-αlevel detection by ELISA.This study indicated that the secretion of serum ALT decreased due to the transfusion of RAW264.7- Tim-4 compared with corresponding control cells; HE staining and TUNEL showed hepatic injury was alleviated in the group of RAW264.7- Tim-4 transfusion.In addition,the level of serum TNF-αdecreased. These results suggest that macrophage overexpressing Tim-4 molecules prevent mouse from acute hepatic injury induced by ConA.It is consistent with in vitro results.Since this model imitates human virus hepatitis and autoimmune liver disease very well,it is possible to treat this kind of disease with Tim-4 molecules. But concrete molecular mechanism and signal pathway should be futher clarified in the future. Partâ…¡STUDY ON THE RELATIONSHIP BETWEEN SINGLE NUCLEOTIDE POLYMORPHISM OF TRAIL GENE PROMOTER REGION AND FATTY LIVER DISEASE IN A CHINESE HAN POPULATION FROM SHANDONG PROVINCEFatty liver disease is a status that lipid accumulates in the hepatocytes and is one of commonly diffuse hepatic diseases,especially in non- or alcoholic fatty liver disease and chronic viral hepatitis infected by HCV.FLD is not a benign and resting pathological status,but an early pathologic changes of hepatic fibrosis,cirrhosis or cancer.Epidemiologic data showed incidence of FLD is 5.2%～11.4%in China and FLD patients are much younger than before. However,pathogenesis remains to be uninvestigated to date.Growing data show FLD is associated with genetic and environmental factor in non- or alcoholic fatty liver disease,and is a common disease of multifactorial inheritance. Therefore,studies of sensitive genes are helpful to elucidate pathogenesis and diagnosis or therapy for FLD.TRAIL(tumor necrosis factor-related apoptosis-inducing ligand) is a newly identified and cloned member of TNF(tumor necrosis factor) by Wiley in 1995. Recent studies showed that expression of TRAIL increased in viral hepatitis and after alcohol intake,which accelerated generation and development of diseases. So TRAIL gene may act as a potentially sensitive gene in human fatty liver disease.Our previous study showed that SNPs of TRAIL at 1525G/A and 1595C/T in 3'-UTR region were related to development of FLD in Chinese Han populations.Alexandra Weber and his colleague found there were four SNPs (-707C/T,-665T/C,-621C/T and -597A/G) within TRAIL gene promoter region in Europe population in 2004.Therefore,this study is aimed to assess the relationship between the four SNPs and non- or alcoholic fatty liver disease susceptibility by a case-control study in a Chinese Han population.In this study,109 fatty liver disease patients(including 42 non- or 67 alcoholic) and 132 healthy controls were recruited.DNA extracted from their peripheral blood were taken as research samples.Four SNPs reported by Alexandra Weber were detected with polymerase chain reaction and restriction fragment length polymorphism(PCR-RFLP) and Tetra-primer ARMS-PCR method in Chinese Han population from Shandong Province.However,no SNPs at -707,-665,-597 sites were detected in Chinese Han population from Shandong Province; Frequencies of CC/CT/TT genotypes at -621 site were 0.238/0.500/0.262, 0.269/0.448/0.284 and 0.280/0.394/0.326 in the non- or alcoholic fatty liver disease patients and healthy controls.But the site showed no significant difference in the different groups.The polymorphisms in this study showed no association with FLD susceptibility in a Chinese Han population from Shandong Province.The role of polymorphisms in the promoter region of TRAIL gene to pathogenesis of FLD should be further clarified by enlarging samples and analyzing more different groups.