Experimental Study Of GM-CSF Gene Therapy Of Leukemia

The leukemia is a common malignant tumor for children and teenagers. The treatments of leukemia were confined by the diffusion of leukemia cells. Tumor immunity is primarily cell-mediated, with T lymphocytes as the main effector cells. Tumor antigens are first processed and presented by antigen presenting cells (APC). The immunogenic peptides are expressed on APC in the context of MHC class I molecules and presented to T lymphocytes. Dendritic cell (DC) is the most important APC in the induction of anti-tumor immune response. GM-CSF is an important immune regulating cytokine that promote maturation and differentiation of DC, up-regulate their expression of MHC molecules and costimulatory molecules, induce proliferation and differentiation of granulocyte and monocyte-macrophage lineages. In the present study, a mouse GM-CSF gene recombinant eukaryotic expressing plasmid was constructed. The recombinant was transfected into FBL-3 cells by electroporation. The aim of this study is to investigate efficiency of vaccination with mitomycin-C inactivated FBL-3-GM-CSF into mice and feasibility of immune therapy of leukemia. The main results are as follows: 1. The construction of pcDNA3-GM-CSF eukaryotic expressing plasmidMouse GM-CSF cDNA was amplified from the recombinant of PET-30a ( + ) -GM-CSF prokaryotic expressing plasmid by PCR first and BamH I and EcoR I restriction sites were introduced. The inserted fragment was cut by BamH I and EcoR I digestion and ligated into pcDNA3 vector. The pcDNA3-GM-CSF eukaryotic expressing plasmid was constructed. The recombinant was cleaved with appropriate endoneucleases and sequenced. The results showed that the orientation of the insert was found to be correct , while no rearrangement or mutation was found. 2. Transfection into FBL-3 cells by electroporation and selection ofthe high GM-CSF expressing cell cloneThe pcDNA3-GM-CSF eukaryotic expressing plasmid was transfected into FBL-3 cells by electroporation. After screening by G4i8 and cloning by limiting dilution, we obtained positive cell clones (FBL-3-GM-CSF). PCR and RT-PCR assay showed that GM-CSF gene has integrated into FBL-3-GM-CSF cells and stably expressed. Then, a high GM-CSF expressing cell clone (clone 3) was selected by RT-PCR, hematopoietic progenitor cell proliferative assay and hematopoietic progenitor cell colony formation assay. 3.The biological characteristics of FBL-3-GM-CSF cellsThe biological characteristics of FBL-3-GM-CSF cells hadn't obviously difference comparing with FBL-3-vect and FBL-3 cells, including cellular morphology and growth rates and so on.The FBL-3-GM- CSF cells were injected into the syngenic mice and found that tumor formation was delayed and time of survival was lengthened slightly as compared to the FBL-3-vect and FBL-3 cells. 4.Efficiency of vaccination with mitomycin-C inactivated FBL-3-GM-CSF into mice and feasibility of immune therapy of leukemiaThe date of animal experiments showed that vaccination with mitomycin-C inactivated FBL-3-GM-CSF into mice could induce potent anti-tumor immune reaction compared with FBL-3- vect, FBL-3 cells and PBS. For vaccination with mitomycin-C inactivated FBL-3-GM-CSF into mice, the growth rates of tumor were decreased markedly and time of survival of mice was lengthened dramatically as compared to the FBL-3-vect and FBL-3 cells and PBS groups. The above results showed efficiency of vaccination with mitomycin-C inactivated FBL-3-GM-CSF into mice and feasibility of immune therapy of leukemia. The study provided experimental and theoretical data for clinical study and application.