Experimental Study On DcR3 To Prevent Liver Allograft From Damage After Transplantation

Decoy receptor 3 (DcR3) is an immunosuppressive molecule which was found recently, belongs to the tumor necrosis factor receptor (TNFR) family. The open reading frame of DcR3 encodes 300 amino acids with a 29-residue signal sequence, but-' without transmembrane region, so it is a secreted protein. The DcR3 expression is detected in stomach, spinal cord, colon, lymph node, spleen, endothelial cells, as well as carcinoma cell lines derived from colon and lung carcinoma. DcR3 is known to bind to three TNF family ligands, namely Fas ligand (FasL), LIGHT, and TLIA.Many studies have been showed that DcR3 mRNA and protein overexpress in human gastrointestinal carcinoma, hepatocellular carcinoma, pancreatic carcinoma, lymphoma, malignant glioma and glioblastoma. DcR3 expressed by carcinoma cells can favor carcinoma cells to escape immune surveillance, prevent cytotoxic effects of CTL and accelerate the progress of malignant tumor.Recent investigations have been showed a compromised insulin release and an increased apoptosis among islets that are treated with FasL and gamma-interferon (IFN-gamma) in combination. DcR3 significantly reduces such apoptosis, prevents such compromise, increases success rate of islet transplantation and prolongs survival time of islet allograft. Some studies showed intravenous administration of DcR3 protein daily for seven days starting from one day before heart transplantation could prolong the mean survival time of heart allograft of mice for 3.3 days. These results indicate that immunosuppressive effects of DcR3 may play an important role in the repressing of graft rejection.DcR3 blocks FasL mediated apoptosis by competing with Fas and binding to FasL. DcR3 also plays immunosuppressive effects by regulating the interaction of T lymphocyte with antigen presenting cell, modulating differentiation and activation of macrophage and dentritic cell, inhibiting T cell expansion in immune response, suppressing T cell chemotaxis toward CXCL12/SDF-1 α and FasL. In connection with liver transplantation,DcR3 may act as a decoy receptor by suppressing T cell chemotaxis toward CXCL12/SDF-1 α and FasL, preventing the attack of CTL through a Fas/FasL pathway. However, intravenous administration of DcR3 protein has systemic immunosuppressive effects. Therefore, we speculate that expression of DcR3 in liver allograft by transfection may be helpful to protect liver allograft from the attacks of CTL.The genome of AAV can be integrated into chromosome at a fixed point. It can avoid insert mutation caused by randomly integration and ensure long-term steady expression of target gene. Thus AAV can be used as an ideal vector of immune-modulating or graft-protective molecules to induce liver transplantation tolerance. Because DcR3 protein is metabolized rapidly and its half-time is about 20 minutes, we can use AAV as a vector of DcR3 to infect liver allograft, and gain long-term expression of DcR3 protein in liver allograft and protective effects to liver allograft. At the same time, it can avoid systemic side-effects caused by intravenous administration of DcR3 protein.To confirm our hypothesis, we plan to express DcR3 in liver allograft by transfection, and take the advantage of its immunosuppressive effects to prolong the survival time of liver allograft by inhibiting the attack of CTL. We cloned DcR3 gene, constructed and identified recombinant plasmid and DcR3/AAV. We then established inbred rat liver transplantation model and investigated the protective effects of DcR3 to liver allograft. Through these experiments, we wanted to clarify the protective effect of DcR3 to liver allograft, and explore a novel measure of preventing allograft rejection and prolonging the survival time of liver allograft.The results of our researches include:Firstly, DcR3 cDNA was cloned by PCR and inserted into pAAV-IRES-hrGFP plasmid. We identified the recombinants of DcR3/pAAV-IRES-hrGFP plasmid in eukaryotic cells and gained correct DcR3 expression in the recombinant DcR3/pAAV-IRES-hrGFP plasmid which provided a basis for construction of recombinant DcR3/AAV. We also detected the expressing products of recombinant DcR3/pAAV-IRES-hrGFP plasmid in eukaryotic cells by confocus microscopy and demonstrated the concordance of GFP and DcR3 expressing in eukaryotic cells. It indicates that GFP expressing in eukaryotic cells which were transfected by recombinant plasmid could act as a sign of DcR3 expressing in eukaryotic cells.Secondly, we successfully constructed and purified recombinant DcR3/AAV byoptimizing the conditions of transfecting AAV-293 cells. Then we measured the purity and titre of recombinant DcR3/AAV. We furtherly detected the expression of DcR3 by infecting eukaryotic cells with purified recombinant DcR3/AAV and gained correct DcR3 expression. Thirdly, we established inbred rat liver transplantation model and infected liver allograft with recombinant DcR3/AAV. Then we observed the survival time, DcR3 expression and pathological changes in liver allograft, and measured the changes of hepatic function. The results indicate that DcR3 expression is correlated with the changes of hepatic function, delayed lymphocytes infiltration, decreased number of lymphocytes mfiltratedinto liver allograft and mild pathological changes in liver allograft. There is no expression of transfected DcR3 gene in the other organs all the time.In conclusion, our results demonstrate that transfected DcR3 gene expressed only in liver. DcR3 expression can prolong the survival time of recipient rat, relieve the injuries to hepatic function, delay lymphocytes infiltration and decrease the number of lymphocytes infiltrating into liver allograft, lessen the hepatic tissue destruction caused by infiltrating lymphocytes. Therefore, DcR3 can protect liver allograft from graft rejection and prolong the survival time of liver allograft, so it has a therapeutic potential in graft rejection.