| Background:Liver transplantation was the best choice of patients in the terminal stage. However, the rejection of recipient to the transplanted organ, which depends on T-cell activation and proliferation was the major problem for the success of the liver transplantation. In recent research, it was found that two distinct signals were required for full T-cell activation. The first signal was evoked by the T-cell receptor after engagement with the major histocompatibility complex-peptide complex expressed on the surface of antigen-presenting cells (APCs). The second costimulatory signal was provided by T-cell surface receptors (CD28) after binding to their ligands, B7 on the surface of APCs. Costimulatory molecules has the function to prime, sustain and activation cascade reaction, which decide the cell to proliferate, to be inhibited or to became anergy, even apotosis. T cells couldn't differentiate nor proliferate, not to say causing immune response to transplanted organ in the absent of any one of the costimulator molecules. The rejection caused by T cells needs to identify special antigen and costimulatory signal induced by cell adhesion molecule. Costimulatory signal major depended on the expression of costimulatory molecule on APCs to influence of costimulatory signal on immune rejection. The blockage of costimulatory signal was proved to be an effective way to inhibit the rejection in the organ transplantation because it was hard to control the antigen in donator organ.The superfamily of B7 and their receptors are type I transmembrane glycoprotein, they belonged to immunoglobulin superfamily. There are about 20%-35% amino acid sequence was same in the superfamily of B7. B7-1 (CD80) and B7-2 (CD86) are costimulatory molecules, their receptors conclude CD28,CTLA-4,ICOS,PD-1 and B and T lymphocyte attenuator (BTLA). All these costimulatory molecules not only support the growth and the differentiation of T-cells, but also provide the negative signal to inhibit the immune response the T-cells, like CTLA-4,PD-1 and BTLA. CTLA-4 could blockage the cell cycle to G1 phase to inhibit the activation of T-cell by decreasing the expression of IL-2 and IL-2R.CTLA-4Ig was the gene recombination fusion protein of cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) and IgG Fc, and it was first produced by Linsley et al in 1991. As Linsley reported, CTLA-4Ig could block the costimulatory passageway, which was necessary in the T-cell activated process. And it played an important part in the organ-graft refection.In the exprement, CTLA-4Ig was used to block the second signal in cell immune response caused by acute rejection in orthotopic liver transplantation in rats (ROLT). Immunohisto chemistry and RT-PCR were used to detect the expression of B7-1 and B7-2. The relationship of the costimulatory molecule B7 expression and the acute immunological rejection in orthotopic liver transplantation in rats was researched. However, there are no such reports or papers on the studies as far as we known.Objectives: To observe the effect of CTLA-4Ig on preventing acute rejection and the role of costimulatory molecule B7-1 and B7-2 via the model of orthotopic liver transplantation (ROLT) in rats.Methods:1. Forty-eight adult male DA rats (180 g to 200 g) originally purchased from the animal experiment center of Harbin University which were maintained under conventional conditions. Forty-eight adult male Lewis rats (160 g to 200 g) originally purchased from Beijing Weitonglihua animal experiment limited company which were maintained under conventional conditions. Rats had free access to standard chow and tap water. The rats were fasted for 24 hours before the surgery. All rats were divided into 2 groups randomly.:Group A was control group. Group B was injected with CTLA4-Ig into abdominal cavity after 48 hours of operation.2. Build the model of ROLT (DA- Lewis). All rats were divided into 2 groups randomly.. Group A was control group. Group B was injected with CTLA4-Ig into abdominal cavity after 48 hours of operation. Animals were sacrificed on the day 3,5,7,10 after ROLT to detect the changes of serum biochemistry (ALT,TBIL and DBEL).Liver grafts were collected for measuring the expression of B7-1 and B7-2 mRNA by RT-PCR.3. To observe general state of rats health.4. Animals were sacrificed on the day 3,5,7,10 after ROLT to detect the changes of serum biochemistry(ALT,TBIL and DBEL).5. RT-PCR: The expression of B7-1 and B7-2 mRNA was analyzed by real-time reverse transcription polymerase chain reaction (RT-PCR). The sequence of the upstresm and downstream primers for B7-1, B7-2 andβ-actin were as follows:5'-GGCATTGCTGTCCTGTGATTAC-3' and 5'-ACTCAGTTATGTTGG GGGTAGG-3' for B7-1;5'-GCTCGTAGTATTTTGGCAGGACC-3' and 5'-CGGG TATCCTTGCTTAGATGAGC-3' for B7-2;5'-ATGGARTCCTGTGGCATCCA-3' ,and5'-CGCTCAGGAGGAGCAATGAT-3' forβ-actin respectively. The product size for B7-1, B7-2 andβ-actin were 314bp, 337bp and 224bp respectively.6. Immunohsitochemistry: The expression of B7-1 and B7-2 protein was analyzed by immunohistochemistriy. SABC measure was used to analysis the relationship between I/R injury and the expression of B7-1 and B7-2 protein in rats' liver. At the same time observe where B7-1 and B7-2 protein are expressed.Results:1. In group B, no clinical rejection character were obserbed(P<0.01).2. In group B, serum biochemistry was lower than cortol group (P<0.01).3. Compared with the group B, the expression of B7-1 and B7-2 mRNA increased significantly in the rats of control group (p<0.01).4. Compared with the group B, the expression of B7-1 and B7-2 protein increased significantly in the rats of control group (p<0.01). Conclusions:1. To block the costimulatory molecule B7-1 and B7-2 might be the primary target to prevent early rejection after liver transplantation.2. Acute rejection may increase the immunogenticity of the liver by upregulating the costimulatory molecule B7-1 and B7-2.3. CTLA-4Ig treatment can abate the expression of B7-1 and B7-2mRNA.4. The dynami determination of costimulatory molecule B7 is helpful to the observation of rejection course in rats.
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