| Lung cancer is one of the most common malignant tumors in the world,and its mortality rate ranks first among cancer deaths.According to World Health Organization statistics,about 1.76 million people die of lung cancer each year.The vast majority of patients with lung cancer at the time of clinical diagnosis are mostly advanced or incurable stage IIIb or stage IV,so they have lost the chance of radical surgery.In recent years,immunotherapy has gradually been applied clinically,and has shown certain advantages,and the prognosis of lung cancer has improved,so it is recommended as a standard treatment for advanced lung cancer.Among them,tumor-specific T cells are immune cells with good development prospects at present,so the discovery of target antigens that can be recognized by T lymphocytes is the key to treatment.The cancer testis antigen(CTA)family is an important tumor-associated antigen in lung cancer tissues.The expression pattern of the cancer testis antigen family has a certain degree of specificity: members of this protein family are expressed in a variety of cancers,such as lung cancer,breast cancer,and melanoma.For normal tissues,the cancer testis antigen is only in the testis It is expressed in cells and occasionally can be detected in placental tissue,and the testes have immunity immunity,and will not be recognized and killed by the immune system.Therefore,the cancer testis antigen family is an ideal target for tumor immunotherapy.Melanoma antigen gene(MAGE)is one of the subfamilies of cancer testis antigen family,which is a tumor-associated antigen,and it has immunogenicity and restricted expression.According to research,the protein encoded by the MAGE family is tumor rejection antigen(TRA),which can be processed in cells to form tumor rejection antigen peptide(TRA Peptide)and interact with human leukocyte antigen(HLA).Combine to form a complex that can induce cytotoxic T lymphocytes(CTLs)with specific killing ability to tumor cells.The expression pattern of the MAGE family is the same as that of the CTA family,and it is expressed in a variety of tumor cells,such as breast cancer,melanoma,esophageal cancer,and colorectal cancer,and is not expressed in most normal tissues.This study believes that the MAGE family has great potential as a candidate target for CTL-mediated tumor-specific immunotherapy.The in-depth research and understanding of the MAGE family is helpful for molecular biology diagnosis and personalized molecular targeted therapy of lung cancer.Dendritie cells(DC)are one of the non-mononuclear phagocytic cells.They are the most powerful antigen-presenting cells(APC)known in the human body.The generation of anti-tumor immunity depends on DC The cells capture and process tumor antigens,and then present the antigen information to immature T cells in lymphoid tissues and activate the body's specific anti-tumor immune response.Tumor-specific cytotoxic T lymphocytes(CTLs)have strong killing ability and strong proliferative activity.This study intends to co-culture and induce tumor-specific CTL cells with MAGE-A10 antigen peptide and DC cells to effectively kill cancer cell lines.Some scholars have conducted similar studies using a common epitope peptide similar to the gene sequence of the MAGE family,and proved that this short peptide has the killing ability to tumor cells.Anti-tumor immunotherapy is not only limited by the immunogenicity of the surface of tumor cells,but also by the amount and rate of expression of tumor-specific antigens.The expression of target antigen on the surface of tumor cells is the core of peptide-based immunotherapy.Increasing the expression of target antigen can improve the effect of tumor immunotherapy to a certain extent.5-aza-2'-deoxycytidine(DAC)is a specific methylation inhibitor,which can reduce the level of methylation,and the drug can increase the expression of MAGE-A10.Therefore,in the present study,DAC was used to treat lung cancer cell lines in the hope that it would induce increased expression of MAGE-A10.In lung cancer,the relationship between the high expression of MAGE-A10 and the clinicopathological parameters of patients and the survival of patients is inconclusive.Moreover,there have been no reports on the use of MAGE-A10 antigen peptide and demethylation reagent DAC immunotherapy for immunotherapy of lung cancer.Therefore,we studied the killing effect of MAGE-A10 antigen peptide combined with the demethylation reagent on lung cancer in vitro.The expression of MAGE-A10 at the protein level in lung cancer tissues was detected by immunohistochemistry,and the relationship between the expression of MAGE-A10 and the clinicopathological characteristics of patients was analyzed.Survival analysis was performed by Kaplan-Meier Plotter online public database to verify the effect of lung cancer patients on prognosis.It was verified in different lung cancer primary cells and cell lines whether the demethylation reagent DAC can induce the increase of MAGE-A10 expression on the surface of lung cancer cells and the killing effect of MAGE-A10 specific CTL on lung cancer cells induced by demethylation reagent.Part one Expression of MAGE-A10 Antigen in Human Lung Cancer and Its Clinical SignificanceObjective: Immunohistochemical method was used to study the expression of MAGE-A10 antigen in the tissues of patients with lung cancer and its correlation with clinicopathological parameters,and to establish a theoretical basis for MAGE-A10 antigen peptides to participate in tumor immunotherapy.Methods: 1.Immunohistochemical method was used to detect the expression of MAGE-A10 antigen in normal lung tissue and lung cancer patients.2.The correlation between MAGE-A10 antigen expression and clinical pathological parameters of patients was analyzed.3.Kaplan-Meier survival analysis was used to analyze the positive expression of MAGE-A10 antigen and the 5-year overall survival rate of patients with lung cancer.Results: 1.Immunohistochemical results showed that MAGE-A10 protein was rarely expressed in normal lung tissues in 4 cases,negative in 26 cases,with a positive expression rate of 13.3%,positive expression in lung cancer tissues,positive expression in 198 cases,and negative expression.In 218 cases,the positive expression rate was 47.6%,which was expressed in the cytoplasm and nucleus of lung cancer.2.The statistical results showed that the expression of MAGE-A10 protein had no correlation with the age,sex,tumor size,clinical stage,and clinical stage of lung cancer patients(P> 0.05),but the nodal metastasis(6.81,0.0090)and recurrent metastasis(4.64,0.0313)were correlated(P <0.05).3.The 5-year mortality of MAGE-A10 positive expression patients was higher than that of negative expression patients,but there was no significant difference in overall survival rate.Log Rank test result P = 5.002e-10.Conclusion: MAGE-A10 antigen is positively expressed in lung cancer tissues and rarely expressed in normal lung tissues,suggesting that MAGE-A10 antigen can be used as a target antigen for immunotherapy of lung cancer.MAGE-A10 may be associated with poor prognosis in patients with lung cancer.Part two Expression of MAGE-A10 Antigen in Human Lung Cancer Cell Lines and Primary Cells and Bioinformatics Analysis of MAGE-A10Objective: To study the expression of MAGE-A10 in human lung cancer cell lines and primary cells;use bioinformatics to analyze the relationship between MAGE-A10 expression and patient survival.Methods:1.The expression of MAGE-A10 was detected in human lung cancer cell lines A549 and H1975 and primary cells L228,L419 and L329 by q RT-PCR and Western blot.2.1926 OS samples(344 FP samples and 982 PPS samples)were obtained from Kaplan-Meier Plotter online public database(http://www.kmplot.com),and were conducted survival analysis.Results: 1.The expression of MAGE-A10 m RNA in A549 cells was at low levels and at high levels in H1975 cells.In primary lung cancer cells,the expression of MAGE-A10 m RNA in L228 and L419 cells were at high levels,and at low levels in L329 cells.2.Based on the analysis of the open public database Kaplan-Meier Plotter,the overall survival,post-progression survival,and first progression time of lung cancer patients with high expression of MAGE-A10 are shorter than those of patients with low expression of MAGE-A10(P < 0.05).Conclusion:1.The expression of MAGE-A10 m RNA was at high levels in H1975,L228 and L419 cells,and was at low levels in A549 and L329 cells.2.The lung cancer patients with high expression of MAGE-A10 have shorter overall survival,post-progressive survival and first progression time than those with low expression of MAGE-A10.Part three Study on the effect of demethylated reagent on the killing activity of MAGE-A10 antigen peptide specific CTL and its killing effect on lung cancer cellsObjective:To study the effect of demethylation reagent DAC on the killing activity of MAGE-A10 antigen peptide specific CTL and its killing effect on lung cancer cells.Methods:1.The expression of MAGE-A10 m RNA and protein were detected in lung cancer cells H1975,L228,L419,A549 and L329 cells treated with the demethylation reagent DAC by q RT-PCR and Western blot.2.ELISA method was used to detect the IFN-? secretion level in the culture supernatant of MAGE-A10 common antigen-specific CTL mixed with primary cells of lung cancer tissues.3.Cytotoxicity test was use to detect the effect of MAGE-A10 antigen peptide specific CTL on the killing effect of primary cells in lung cancer tissues.Results: 1.Demethylation reagent DAC can induce the increase of MAGE-A10 expression in H1975 cells,L228 cells and L419 cells,and the expression of MAGE-A10 protein and m RNA are dose-dependent.2.The results of IFN-? release experiments showed that the IFN-? secretion level of the DAC group was significantly increased when MAGE-A10 antigen peptide and DAC were added simultaneously,and the higher the target-effect ratio,the higher the IFN-? secretion level(P <0.05).3.The results of cytotoxicity experiments showed that the MAGE-A10 antigen peptide + DAC group had the highest killing efficiency under the same effect target ratio.The killing efficiency is highest when the effective target ratio is 40: 1.Conclusions:1.Demethylation reagent DAC can increase the expression of MAGEA10 in H1975 cells,L228 cells and L419 cells.2.Demethylation reagent DAC can enhance the killing function of MAGE-A10 common antigen peptide specific CTL on H1975 cells,L228 cells and L419 cells. |
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