Blockade Of Icos Costimulatory Pathway To Prevent Acute Rejection In Rat Liver Transplantation

PARTⅠ CONSTRUCTION AND IDENTIFICATION OF EXPRESSING SIRNA PLASMID AGAINST RAT ICOS Objective: To successfully develop a king of RNA interference (RNAi) that specifically targets rat Inducible Costimulatory Molecule (ICOS) by synthesizing small interfering RNA (siRNA) in vivo, and to assess the inhibitory effect of this siRNA on ICOS expression of rat activated T lymphocytes. Methods: According to mechanism of RNA interference, the interferential recombinant plasmid pICOS-A targeting rat T lymphocytes gene sequence and its unrelated control plasmid pICOS-B were constructed based on pGenesil-2 vector, and were identified with restriction-endonuclease digestion and sequencing. The recombinant was transfected into T lymphocytes using Lipofectamine. The reverse transcription PCR (RT-PCR) was performed to detect the expression of ICOS mRNA. Results: The interferential recombinant plasmid pICOS-A and its unrelated control plasmid pICOS-B were successfully constructed identified with restriction-endonuclease digestion and sequencing. RT-PCR showed that pICOS-A inhibited the expression of ICOS mRNA in rat activated T lymphocytes significantly, reduced about 84.5% , while the control plasmid pICOS-B showed no inhibitory effect. Conclusion: The results showed that the small interfering RNA targeting rat ICOS suppressed the expression of ICOS of rat activated T lymphocytes in a sequence-specific manner. PART Ⅱ BLOCKADE OF ICOS COSTIMULATORY PATHWAY TO PREVENT ACUTE REJECTION IN RAT LIVER TRANSPLANTATION Objective: To investigate the effect of blockade of ICOS costimulatory pathway with RNAi in acute rejection of rat orthotopic liver transplantation. Methods: 18 cases of orthotopic liver transplantation were performed in Lewis to BN rats by using "the two-cuff technique",and all rats were divided into interference group, rejection group and contrast group in random, every group had 6 cases. The BN rats of interference group were injected with 2×106 (1 ml) BN rat T lymphocytes which were transfected with interferential recombinant plasmid pICOS-A in advance during operation. In contrast group, the injected BN rat T lymphocytes ( 2×106, 1 ml) were transfected with empty vector pGenesil-2 in advance. While in rejection group, injected with 1 ml isotonic Na chloride. One week survival rate of every group were observed. On the 8th day after transplantation, in each group 3 rats were killed to study for research. Peripheral vena blood was obtained to detect the positive rate of CD25 and ICOS in T lymphocytes respectively by flow cytometry. Hepatic tissue was obtained to study the pathological changes and apoptosis with TUNEL. RT-PCR was performed to detect the expression of ICOS mRNA; meanwhile, the protein level of ICOS was measured with immunocytochemistry and Western blotting. The lymphocytes were obtained from spleen. The mixed lymphocyte reaction (MLR) was performed to observed the stimulation index (SI). Detected IFN-γ and TNF-α levels in plasma with ELISA. Results: One week survival rate of interference group was 83.3%,markedly higher than that of rejection group and contrast group, both were 50% ( P<0.01); There had been serious acute rejection in allograft liver in both rejection and contrast group, but in interference group, it was not visible; Flow cytometry analysis showed that the positive rate of ICOS in peripheral blood T lymphocytes in interference group was 21.3±1.4%, significantly lower than that of rejection and contrast group, which was 39.1±4.6% and 39.1±2.1% respectively ( P<0.01); And the percentage of activated T lymphocytes in total T lymphocytes in interference group was 15.2±0.7%, also lower than that of rejection and contrast group ( P<0.01); Hepatic tissue TUNEL staining showed there were more apoptotic T lymphocytes in interference group (51.3±11.0/mm2) than in rejection group (26.3±4.2/mm2) (P < 0.01) ; RT-PCR, Western blotting and immunocytochemistry all indicated the expression of ICOS gene and protein in allograft livers were suppressed in interference group; MLR a...