Establishment Of HPV16E6Lung Cancer Model And Cultivation Of HPV16E6Antigen Specificity Dendertic Cell

Background and objective:Lung cancer is one of the fastest growing,morbidity and mortality tumors in the world. The complexity of the pathogenesis of lung cancer,smoking is considered to be the main reason. Since Syrjanen first proposed human papillomavirus infection may be related to lung cancer, many studies have shown the relation between HPV infection and lung cancer. Further studies showed that HPVE6/E7protein has an important role in promoting lung epithelial cell proliferation and tumorigenicity. Human papillomavirus E6protein (HPVE6) is constitute by151amino acid residues and19,000molecular weight, containing two zinc fingers,and each containing two zinc finger CXXC sequences and having antigenicity. Our previous study found that in lung cancer patients, about52%of the patients can be detected with HPV16E6protein antigens, mainly in cytoplasm and nuclear membrane of lung cancer cells. Also,we found that lung cancer with HPVE6protein antigen expression was significantly increased infiltration of lymphocytes around. Foreign study also found that in patients with HPV16-positive lung cancer patients expressing E6-associated protein, especially11to20marked by the expression, often have increased T cell reactivity. Therefore, we believe that HPV16E6protein may be a common lung cancer-specific tumor antigen. With HPV16E6antigen-specific T lymphocytes adoptive immunotherapy, may be obtained encouraging efficacy in lung cancer patients with positive HPV16E6body, thus changing the current predicament of lung cancer-specific lymphocytes for adoptive therapy.Dendritic cells are the body’s most powerful antigen-presenting cells and induce body generate antigen-specific cytotoxic T lymphocyte response (cytotoxic T lymphocyte, CTL response), they have an important role in our body’s anti-tumor immunotherapy. Studies have shown that tumor-associated antigen sensitized DC can produce a strong induction of T cell function in vitro by culturing DC, and with tumor lysate and tumor apoptosis cells sensitized DC transplantation in vivo and induce the patient to produce specific T cell immune killing tumor cells to arouse anti-tumor immune response. Umbilical cord blood, bone marrow and peripheral blood are the major source of human dendritic cell cultures, In clinical, many sources of DC using peripheral blood mononuclear cells for its yield, high purity and easy to obtain. Peripheral blood mononuclear cells were cultured into DC and reinfusion in patients after sensitization in vivo to induce anti-tumor treatment and also achieved a certain effect. In the early, dendritic cells most derived from bone marrow hematopoietic stem cells, later the researchers found that eripheral blood mononuclear cells in the induction of cytokines GM-CSF and IL-4can obtain immature DC.Meanwhile stimulated by tumor antigen or the tumor necrosis factor (TNF-a) can obtained mature DC.In this study, the model built HPV16E6lung cancer cells, and by cultured tumor antigen-specific dendritic cells to lay the foundation for the later verification of tumor antigen-specific T lymphocytes and therapeutic efficacy in animal models.Experimental Methods:1. Synthetic HPV16E6gene and connected the HPV16E6gene to pEGFP-N1plasmid vector, construct capable of expressing green fluorescent recombinant plasmid, and re-extracted plasmid DNA.2. In order to build HPV16E6lung cancer cell model,use slow virus package recombinant plasmid that express HPV16E6gene and infected H1299lung carcinoma cells with the supernatant.3. Extracted peripheral blood mononuclear cells after culture and tumor antigen-specific tumor antigen stimulation to build tumor antigen-specific dendritic cells.Experimental results:1. Successfully constructed a plasmid vector containing HPV16E6gene, after double digestion of plasmid and PCR amplification,obtain approximately500bp gene fragment.2. Use slow virus package recombinant plasmid that express HPV16E6gene and successfully infected H1299lung carcinoma cells with the supernatant. Further more,successfully build HPV16E6lung cancer cell model. Extracted cell RNA, reverse transcription, and RT-PCR, successfully verified HPV16E6gene overexpression in H1299lung cancer cells.3. This experiment extracted from peripheral blood mononuclear cells, cytokine added to stimulate dendritic cells mature,Detect phenotype by flow cytometry after stimulation of dendritic cells with tumor-specific antigens or not. The former higher express cell phenotype CD80and CD86, a higher degree of maturation. The experiments show that by HPV16E6antigenic stimulation can promote DCs to mature and lay the foundation for the preparation of HPV16E6specific T lymphocytes.