Cloning Of Murine CD40 Ligand CDNA And Preliminary Study Of Murine CD40 Ligand Gene Therapy Of Mouse Hepatocellular Carcinoma

Backround and Objective:CD40 ligand (CD40L) is a member of the tumor necrosis factor family, which is expression on activated T cells and binds to CD40 present on the membrane of antigen-presenting cells(APC). CD40-CD40L interaction plays a crucial role in the activation of APC and in the initiation of both humoral and cellular immune responses. Thus, gene transfer of CD40L has been proposed as an efficient means to treat malignancies. Our study arms at cloning of murine CD40 ligand cDNA, construction of eukaryotic expression recombinant and preliminary study of murine CD40 ligand gene therapy of mouse hepatocellular carcinoma.Material ans Methods:1. We examined the expression of CD40 in human hepatocellular carcinoma(Hcc) tissues by SABC immunohistochemistry. 2. Total cellular RNA was extracted from the Balb/c mouse spleen ceils. Synthesis and amplification of mCD40L cDNA was done using the one-step reverse transcriptase polymerase chain reaction (RT-PCR) system with specific primers and conditions. 3. The mCD40L cDNA fragment directly linked up with T vector to become middle recombinant pUCm-T-mCD40LcDNA. After restriction endonuclease Nhel and EcoRI double digested it, the target fragment was subcloned into eukaryotic vector pcDNA3.1+ to construct eukaryotic expression recombinant pcDNA3.1+-mCD40L cDNA; 4. Building of eukaryotic expression cell strain through that eukaryotic expression recombinant pcDNA3.1+-mCD40L cDNA being transfected H22 cell by lipofectin ad the cell line screened using geneticin(G418).5. Parental H22 or transfectants (H22-CD40L) were inoculated subcutaneously into the left flanks of syngeneic mice(Balb/c) and monitored the tumor growth every week.Result: 1.40% of human HCC tissues demonstrated positive staining for CD40. 2.RT-PCR analysis showed that 0.8kb fragments corresponding to the mCD40LcDNA were amplified. 3.The middle recombinant and expression recombinant were checked by restriction endonuclease digestion and there were confirmed by DNA sequencing respectivelyAThe monoclone cell strain was picked up when transfected H22 cell by lipofectine and the cell was screened with G418 18 days.Through certifying with RT-PCR, eukaryotic stable expression cell strain containing pcDNA3.1+-mCD40L cDNA gene was produced. 5.All syngeneic Balb/c mice inoculated with H22 cells developed subcutaneous tumors, but inoculated of H22-CD40L cells into syngeneic Balb/c mice resulted in completed rejection.Conclusion: 1.40% human HCC tissues have CD40 expression. 2.It has completed the cloning of mouse CD40 ligand cDNA and the construction of eukaryotic expression recombinant pcDNA3.1+-mCD40L cDNA. 3.It was accomplished to set a stable transfective cell strain. 4.The tumorigenicity of H22 cells was abolished by in vitro transfection with mCD40L gene in syngeneic mice.