Detection Of MAGE-A3 Antigen And HLA-class I Gene Distribution In Lung Cancer

For a long time, most of doctors and researchers have been puzzled of the therapy of malignant tumors. These three kinds of traditional therapeutic can't eradicate tumor completely and has its disadvantage respectively.With the development of molecular biology and gene engineering technique, bio-therapy gradually become a promising treatment. Immunotherapy as a part of comprehensive treatment to tumors has a tremendous development after the founding of tumor rejective antigens. The human MAGE genes are expressed in a wide variety of tumors but silent in normal adult tissues except in the male germ cells, placenta, and possibly, cells of the developing embryo. Hence, they encode strictly tumor-specific antigens. This antigens can be presented by human leukocyte antigen class I molecules (HLA-class I molecules) and recognized by autologous cytotoxic T lymphocytes (CTLs), then stimulate the later's reproduction and activation. That represents attractive targets for tumor immunotherapy. MAGE-A3 gene is a member of MAGE gene family. It has high expression in different tumors and different pathological types.Now, the report about MAGE-A3 gene expression is focus on it's mRNA level. But we all know mRNA doesn't mean the expression of MAGE-A3 antigen. Only the protein expression can induce the specific CTLs to recognize and kill MAGE-A3 antigen positive tumor cells. Our lab found that the mRNA of MAGE-A3 has high ratio in lung cancer tissues (about 84%). This suggested that the antigen of MAGE-A3 also probably have high expression in lung cancer. In order to identify this hypothesis, wedetected the positive expression of MAGE-A3 antigen in lung cancer tissues by means of western blot analysis.There are a lot of antigenic determinant (epitope) in MAGE-A3 antigenic protein. They can be presented by different HLA-class I molecules, and recognized by CTLs. Then the later performed it's function to kill the tumor cells expressing MAGE-A3 antigen effectively. So the success of specific immunotherapy mainly determined by HLA-class I molecule. The T cell receptor (TCR) of effective T cells can bind with the complex of HLA-class I molecule and MAGE-A3 related peptide. Only TCR-HLA-class I molecule-MAGE-A3 epitope complex can activate CTLs effectively. With the knowledge of MAGE-A3 antigenic protein expression and HLA-class I distribution in lung cancer tissues we can primarily estimate the ratio of lung cancer patients who can be treated with specific immunotherpay based on MAGE-A3 antigenic peptide. So we detected the distribution of HLA-A1, HLA-A2 and HLA-A24 in lung cancer patients. Methods and materials:1. Samples collection: 70 samples of tumor tissues and 72 samples of tumor adjacent tissues were collected totally. All tissues were frozen in liquid nitorgen immediately after surgery and then stored at - 80℃ until the extraction of protein. In addition 107 samples of normal adult blood (3ml every case) have been collected and Stored at 4℃.2. Tissues protein extraction: Refer to {MOLECULE CLONING)) a laboratory manual, 2nd ed)) . And the protein was stored at -20℃3. DNA extraction: Using the product of Shanghai shenggong Ltd.-UNIQ-10 genome DNA extraction kit. DNA solution was stored at -20℃.4. Western blot analysis: protein samples were separated by 12% sodium dodecyl sulfate polyacrylamide gel eletrophoresis (SDS-PAGE) and transferred to a nitrocellulose membranes. The membrane was blocked with 1% protein powder (wuhanBOSTER Ltd.) in PBS for 2 hours at room temperature or overnight at 4℃, and incubated with primary antiboy -57B(generously presented by Giulio C.Spagnoli M.D.) at room temperature for 2 hours; Protein bands in membrane were stained with a horseradish peroxidase-conjugated secondary antibody-Goat anti Mouse IgG and counterstain with 3'3 diaminobenzidine (DAB) finally. Taking photoes by Bio ImagingSystem, and data or result were kept for statistics and analysis.5. Polymerase chain reaction with sequence specific primer (PCR-SSP): The sequence specifi...