Experimental Study On Immune Tolerance Induced By F Protein In Rats After Orthotopic Liver Transplantation

Objective To explore new targets for transplantation tolerance induced by liver F protein and to investigate the possible mechanism of liver transplant tolerance. Thereafter, our work may provide experimental evidence and theoretical support for successful induction of specific immunological tolerance of human liver transplantation.Methods The rats were randomized into three groups, which included blank control group, immune rejection group and immunological tolerance group. Each group consists of6rats. Orthotopic liver transplantation (OLT) from Wistar rat to SD rat was performed in immune rejection group and immunological tolerance group. The rats from blank control group weren’t performed OLT operation.0.4mg of donor-derived F protein was thymus inoculated on the7th day before transplantation in immunological tolerance group. Wistar rats from blank control group, recipient SD rats from immune rejection group and immunological tolerance group were respectively sacrificed before transplantation, at day7and day100after transplantation. Liver tissues were collected from the three groups. With the protocol of Trizol reagent, total RNA was extracted from liver tissues. Reverse transcription PCR was applied to obtain cDNA of total RNA. Hereafter, detect the expression levels of immunological tolerance-related genes of FLJ11565, MGC45806, DKFZp434K1210and F protein gene with fluorescent quantitative PCR. Apoptosis of infiltrating lymphocytes was evaluated by terminal deoxynucleotidyl transferase-mediated dUTP Nick-end labeling method posttransplantation. Amino acids and small molecules metabolites of hepatic metabonomics were detected with methods of amino acid analysis technique and HPLC-MS technique.Results1. The expression level of immunological tolerance-related genes in immune rejection group was lower than that in blank control group. The expression level in immunological tolerance group was higher than that in immune rejection group and blank control group (P<0.05).2. The apoptosis index of infiltrated lymphocytes in hepatic grafts from blank control group, immune rejection group and immunological tolerance group were (8.83±0.43)%,(11.32±1.29)%and (19.00±1.96)%. respectively. One group was significantly different with any other one (P<0.05).3. Compared with immune rejection group, Taurine, Glutamic acid, Valine, Methionine, Lysine and Tryptophane in immunological tolerance group decreased significantly (P<0.05). While the content of Glycine was obviously higher (P<0.05).47metabolites were discovered by metabolic profiling analysis of liver allograft. The number of elevated metabolites in immune rejection group and immunological tolerance group was5. Compared with blank control group and immune rejection group, the number of increased metabolites in immunological tolerance group was2. And the decreased metabolites were also2. Based on HMDB database searching and structure derivation,18kind of different metabolites were initially identified, including D-Tagatose, Urocanic acid, Etiocholanolone, et al.Conclusion The four immunological tolerance-related genes of FLJ11565, MGC45806, DKFZp434K1210and F protein gene may play an important role in regulatory of immune tolerance. Apoptosis analysis of graft-infiltrating lymphocytes contributes greatly to judging intuitively whether the grafts are in the state of rejection or tolerance. Apoptosis of infiltrating lymphocytes in liver allograft could help to induction of immune tolerance. The immune nutritional amino acids of glutamine, glycine, methionine and so on play an important role in regulatory of immune response in liver transplantation. The combined effects of a variety of immune nutritional amino acids are essential to the induction of immune tolerance after OLT operation. The metabolites of D-tagatose, Urocanic acid and Etiocholanolone. et al. may affect induction of immune tolerance.