The Expression And Preliminary Study Of F Protein In Inducing Immunotolerance

Liver disease may have a serious effect on our health,Hepatocellular carcinoma (HCC)is one of the most prevalent malignant diseases worldwide,It ranks fourth in mortality rate,behind lung,stomach and colon cancers,Virus hepatitis,hepatic fibrosis, hepatocirrhosis and so on are the primary causes of liver cancer.Currently liver transplantation has been regarded as the most benifical measurement for certain end-stage hepatopaty in the word.The use of immunodepressant after liver transplantation can cause serious complications,suah as chronic renal failure,cardiovascular events,onset hypertension and malignant tumors,which greatly affect the patients' life quality.In order to promote the patients' life quality,we should induce immunotolerance on the basis of further optimization and minimization of immunosuppressive therapy after liver transplantation.There are many hypothesis on liver transplantation mechanisms, one of the mechanisms is that F protein can induce immunotolerance.F protein only expressed in liver cell membrane or liver cytoplasma and encoded by loci not linked to the MHC gene.It was reported that this antigen can be detected in sera of almost all liver allotransplanation recipients but its effects remained unknown jet.We use prokaryotic system to express F protein.We expressed and purified the preserved E.coli BL21(DE3)pLysS,and verify it SDS-PAGE and Western blot,the results suggested that the preserved E.coli BL21(DE3)pLysS contained F protein.We expressed E.coli BL21(DE3)pLysS and improved purification condition,and obtained a lot of soluble F protein with low temperature induction.We can obtain higher concentration of F protein using 1/2×Wash buffer,and it can be used to animal experiment.We randomly divided the 20 SD rats into 4 groups,and give them different treatments.The first group was the negative control group,We injected the first groups'thymus with 0.2 milliliter PBS;and injected the second groups'foot-pads with Freund's complete adjuvant which contained 1 milligram F protein,a week later injected them with Freund's incomplete adjuvant which contained 1 milligram F protein,did it every week,and repeated it three times.We injected the third groups'thymus with 4 milligram F protein;the fourth group is the positive control group,we injected their abdominal cavity with cyclophosphamide according to 100 milligram per kilogram.7 days later we put the rats to death,and reserved peripheral blood and thymus.We detected cell apoptosis with TUNEL kit,detected the peripheral blood IL-10 with IL-10 kit.The results indicated that F protein can induce thymic-lymphocytes'apoptosis,and promoted IL-10 secretion.Maybe the mechanism is that when F protein is injected into the thymus,the thymus as a special organ,can generate clonal deletion after it is modified by heterogen antigen.F protein was of important value in early diagnosis and therapy about liver disease,judgement about extent of liver damage,the study on liver transplantation antirejection of immunotolerance.It was difficult to obtain a great quantity of F protein by chemical extract,this prevent other studies' progress.So we obtained rat liver F protein by gene engineering,solved the source problem.Our preliminary study proved F protein's immunogenicity,it may induce immune tolerance after liver transplantation;F protein has immunoreactivity,large dose or repeated low dose can induce recipient antigen specificity lymphocyte apoptosis.Maybe there was also other antigen which participated in liver transplantation' immunoreaction,and determined Transplantation'consequence besides major histocompability complex antigen.