The Research On The Construction Of FasL Plasmid Vector And Its Anti-rejection Effect In Mice Heart Transplantation

In this research project, FasL eukaryotic expressing plasmid vector was constructed. In vitro the FasL gene was transfected into mouse myeloid DC in order to explore the mechanism on inducing T lymphocyte apoptosis. The protective effect to allograft in mouse heart allo-transplantation due to donor derived DCs transfected with FasL was investigated. This study will lay a foundation for advanced FasL gene therapy research of anti-rejection effect.Part I Construction of Eukaryotic Expression Vector of FasLObjective To construct eukaryotic expression vector of mouse FasL in order to prepare transfecting FasL gene into DC. Methods The P43-FasL was cut by restrictive endonuclease-Nhel and NotI to obtain C57 mouse FasL gene which was then connected to pTracer, which had been also cut by restrictive endonuclease-Nhel and NotI. The recombinant vector was transfected to DH5a E coli. for amplification then extraction was performed. At the end identification of the recombinant vector was done by restriction enzyme analysis and the positive clone was sequenced.Results Location of FasL and its sequence in pTracer plasmid is in a right direction by restriction enzyme analysis, and sequence result indicates that clone gene was same with array of C57 mouse FasL of genbank report .Conclusions Eukaryotic expression vector of mouse FasL is successfully constructed and this willmake a foundation to research the biological functions of FasL gene.Part II The experimental research on T lymphocyte apoptosis induced by mouse dendritic cells transfected with FasLObjective To establish the mouse bone marrow-derived dendritic cells with stable FasL protein expression and explore the mechanism of T lymphocyte apoptosis induced by modified DCs in vitro. Methods Mouse myeloid DCs were cultured in selective medium containing necessary cytokines for DC growth in vitro. FasL gene was transfected into DCs with Liposome. The mRNA and protein expression levels of FasL were assayed by real-time quantitative PCR and flowcytometry respectively. The influences on DCs stimulating the proliferation ability of T lymphocyte and inducing its apoptosis were observed through MLC. Results About 1.5-2* 107 myeloid DCs have been harvested from each mouse through cell culture in vitro. After FasL transfected into DC, the FasL mRNA level of DCs is significantly higher than non-transfected DCs groups. The protein expression of FasL in non-transfected DC, empty plasmid transfected DC and FasL transfected DC are 5.59%, 7.96% and 35.77% respectively. The result of MLC demonstrates that the index of stimulation to T lymphocyte of FasL transfected DC significantly decrease, but apoptosis rate of T lymphocyte significantly increases comparing with non-transfected DC and empty plasmid transfected DC(P<0.01).Conclusion Large quantity of myeloid DC with typical histological configuration and high levels of MHCII+ and CDllc+ can obtain through in vitro culture in selective medium which may activate naive T lymphocyte to generate immune response. Transfecting FasL with liposome successfully expresses high levels of FasL protein. The proliferation to T lymphocyte induced by FasL transfected DC becames weaker and its apoptosis is higher.Part III The Research on Anti-rejection Effect due to infusion mouse DCs transfected with FasL in Heart TransplantationObjective To study the protective effect by infusion of the donor bone marrow derived DC transfected by FasL in the graft and its anti-rejection mechanism. Methods At first, the heterotopic heart transplantation model in mice was established, then the experimental animals were divided into 5 subgroups as follows: subgroup 1-treated by DC transfected with FasL, subgroup 2-5 taken as controls: subgroup 2(allograft transplantation), subgroup3 (Normal DC infusion), subgroup 4(isograft transplantation), and subgroup 5(treated with CsA). On the 7th pre-operative day, DCs modified by FasL were transfused into recipients. The graft survivals were individually recorded and pathologically evaluated according to graft rejection grading on the 7 day of post-operation. Furthermore the apoptosis in spleen T lymphocytes induced by FasL transfected DC, empty plasmid transfected DC and non-transfected DC was evaluated by TUNEL method. Results Stable heterotopic heart transplantation model in mice is established successfully, hi subgroup 1-treated by DC transfected with FasL, The Mean Survival Time (MST) of grafts is significantly longer than subgroup2 and subgroup3 (22d vs. 9d, 8d, J°<0.01), accompanied by significantly lower pathological grading of rejection than in subgroups 2 and 3(P<0.01). Through detecting spleen T lymphocyte apoptosis with TUNEL, Apoptosis index (AI) is high in FasL transfected DC ( 11.67±1.53 ) , compared with non-transfected DC ( 2.67±0.58 ) and empty plasmid transfected DC ( 3.33±0.58 ) (PO.01) .Conclusion Mouse heterotopic heart transplantation model is ideal and valuable for transplantation rejection research. In this model, donor-derived DCs transfected with FasL gene could exhibit protective effect on grafts. The anti-rejection mechanism might be due to specially inducing apoptosis of T lymphocyte.