The Study Of Lung Cancer Treated With Vaccine Of Allogenic Tumor Cell Line Transfected Via GM-CSF Gene

Part I:Construction of Tumor Vaccine Using Lung Cancer Cell Line Transfectedwith GM-CSF genePurpose: To construct the tumor vaccine using lung cancer cells lines LLC and LA795 transfected with GM-CSF gene and to identify the gene expression in tumor cells. Methods: Murine GM-CSF gene was amplified by RT-PCR method. The mGM-CSF gene was inserted into pMD18-T vector. The recombinant plasmid was amplified and identified. Amplify the mGM-CSF gene using recombinant plamid as template. The target gene was inserted into pIRES2-EGFP vector to construct mGM-CSF-pIRES2-EGFP plasmid. And this plasmid was used to transfect murine lung cancer cells to prepare tumor vaccine. The level of GM-CSF secreted by the tumor vaccine was tested using ELISA method. Results: The murine GM-CSF gene was amplified correctly by RT-PCR. The recombinant plasmid GM-CSF-pIRES2-EGFP was verified by enzyme digestion and sequencing. Lung cancer cells of mice expressing GM-CSF gene were obtained after the recombinant plasmid GM-CSF-pIRES2-EGFP had been transferred. Transfected lung cancer cells of mice were expressed by corresponding GM-CSF protein after being identified by ELISA method and its quantity of expression is suitable to prepare tumor vaccine. Conclusion: The recombinant plasmid pIRES2-EGFP expressing GM-CSF was constructed successfully. Murine lung cancer cells transfected with GM-CSF gene could secret high level recombinant protein and can be used as tumor vaccine.Prat II: Evaluate the anti-tumor immunity and Mechanism of GM-CSF whole-cellvaccination in animal modelPurpose: To analyze the anti-tumor effects and mechanism of tumor vaccine which was prepared using murine lung cancer cells expressing GM-CSF (autoallergic and allogeneic). Methods: The experimental animals were divided into preventive group, therapy group and chemotherapy group. Preventive group and therapy group were divided into GM-CSF modified tumor vaccine group, autologous and allogeneic tumor vaccine group, PBS control group. Chemotherapy group was divided into GM-CSF autologous and allogeneic tumor vaccine plus chemotherapy group, chemotherapy group. After being preventively inoculated, the mice were inoculated with LLC and then observed tumor growth time and survival time. Changes of serum level of cytokines (IL-4,IFN-γ,TGF- β ) were tested by ELISA method. The phenotype of peripheral blood lymphocytes was tested by flow cytometry method. Test the cytotoxicity of splenocytes against specific tumor cells using LDH method.The percentage of cells activated by specific antigen was analyzed by ELISPOT method. Lymphocyte infiltration of intradermal injection part was analyzed by immunohistochemistry. The therapy group was given different tumor vaccines after inoculation of LLC cells. The chemotherapy group was given small dose of carboplatin chemotherapy and tumor vaccine three days latter. Results: The results showed that the percentage of CD8+T cells in peripheral lymphocytes was increased after inoculation with GM-CSF whole-cell tumor vaccine. ELISOPT experiment showed that the prevalence of activated cells in splenocytes was also increased after the cells were stimulated by specific antigen. The splenocytes and CD8+T cells showed cytotoxicity gainst tumor cells, which indicated that the vaccine could stimulate the specific immune response. Serum level of Thl type cytokines was increased. The tumor growth was inhibited after inoculation with GM-CSF whole-cell tumor vaccines in preventive group. The life span was also longer than control groups. The life span has no significant difference among different therapy groups. Life span of chemotherapy group was longer than that of simple chemotherapy and simple immunity group. The GM-CSF autologous tumor vaccine showed little better effects than allogeneic tumor vaccine, but there was no significant difference. Conclusion: GM-CSF autologous or allogeneic tumor vaccine can stimulate specific immune response against tumor cells. The vaccines can also inhibit tumor growth and prolong the survival time of mice inoculated with tumor cells. The allogeneic tumor vaccine showed great advantage because it is more convenient to construct the vaccine from cell line than autologous tumor tissue. The combination of tumor vaccine and chemotherapy showed favorable prospect.