Anti-cancer Effect Of Specific Cytotoxic T Lymphocytes Activiated By Dendritic Cells Plused With K-ras Antigen To Lung Cancer

Objective: Lung cancer is one of the most common malignant tumors in the world today,and it is also one of the main causes of tumor death in China.Since no obvious clinical symptoms have been found in early lung cancer,about 75% patients have lost the surgery opportunity.Palliative surgery and conventional radiotherapy can not help patients get a good remission rate,so the median survival time of lung cancer patients can only reach 12~18 months.Recently,the biological treatment for lung cancer has been widely used in clinical practice,mainly including immunotherapy and gene therapy.Immunotherapy mainly includes cytokine therapy,immunotherapy and adoptive cellular immunotherapy.Dendritic Cells(DC)is the most powerful antigen presenting cell(APC)in the human body,and the DC treatment of lung cancer has become one of the research highlights in lung cancer.Over the years,a large number of studies have indicated that K-ras gene mutation is an important negative prognostic factor in the development of non small cell lung cancer,and K-ras gene has become an important target in the treatment of lung cancer.In this study,we investigate the killing effect of DC induced by different antigens of CTL induced by different antigens in vivo and in vivo experiments.Methods: DCs were generated from PBMC in the presence of GM-CSF and IL-4 in vitro.DCs were sensitized with lung cancer cell line expressing K-ras mutant,K-ras mutant peptide,K-ras mutant peptide with the surface of cationic nanoparticle.The mature DCs were cocultured with autologous T cells to obtain antigen specific CTL.DC cell surface markers were measured by flow cytometry,MTT assay was used to detect the cytotoxicity of CTL cells to tumor cells in vitro,and the expression level of IL-12 and IFN-was detected by ELISA kit.The anti-cancer activity of CTL in vivo was evaluated in BALB/c nude mice bearing A549(K-ras+)and NCH-446(K-ras-)lung cancer.Results: In vitro experiments,K-ra mutant peptide with the surface of cationic nanoparticle at lower concentrations can be effectively presenting on the surface of DCs than only K-ras mutant peptide.Compared with the single K-ras mutant and the K-ras mutant peptide with cationic nanoparticles,DCs pulsed with whole tumor antigen could better induce CTLs killing activity(P<0.05).The DC with K-ras mutant peptide and K-ras mutant peptide with the surface of cationic nanoparticle could produce specific CTL killing activity against lung cancer cell line A549(K-ras+)(P<0.05),but not NCH-446(K-ras-)(P>0.05).In vivo experiments CTLs cocultured with DCs with the whole tumor antigen significantly inhibited the growth of transplanted pancreatic cancer(A549 and NCH-446)in nude mice.In vivo experiments showed that DCs with K-ras mutant peptide and K-ras mutant peptide with the surface of cationic nanoparticle induced killing activity of specific CTL aganist pancreatic cancer cell line A549(K-ras+)(P<0.05).Conclusion: In vitro,K-ras mutant peptide with cationic carrier can be effectively presenting and expressing of DCs and induce CTL specific killing activity against lung cancer cell lines with K-ras mutant peptide.Moreover,CTL induced with DCs pulsed with K-ras antigen can significantly improve the survival of nude mice with lung cancer,through inhibiting tumor growth.