Study On The Preparation And Anti-tumor Biological Effects Of Gastric Cancer Cell-dendritic Cell Fusion Vaccine

Objective: Gastric cancer is one of the most common malignant tumors in clinicalwork. With the traditional treatments, such as radical operation, chemotherapy andradiotherapy, patients still have to face on a poor prognosis. Dendritic cell is the new focusof anti-tumor biotherapy these years. Many researchers have been attracted by its strongability to present antigen and inspire more effective anti-tumor immune reactions. Gastriccancer cells are lack of differential tumor antigens, with the former methods we only canget few tumor antigens and their ability to irritate dendritc cells or succedent anti-tumorimmune reactions is very tiny. In this experiment, we used cell fusion technology to get thegastric cancer cell-dendritic cell fusion vaccine. We hoped that the fusion cells could getmore tumor antigen information to increase their phenotypes and have more competence toirritate the anti-tumor immune reactions. By this experiment, we hoped to find out whetherthe fusion vaccine has satisfying safety and to verify its ability of inducing anti-tumorimmune reactions. By researching its biological effects, we hoped to get a new kind ofanti-tumor vaccine for gastric cancer's biological therapy.Methods: Part 1. Peripheral blood mononuclear cells were separated from gastriccancer patients and co-cultured with granulocyte-macrophage colony stimulating factors,interleukin-4 and tumor necrosis factor-αto generate mature dendritic cells. The frozenstored human gastric cancer SGC7901 cell lines were resuscitated and cultured at the sametime. The dendritic cells and SGC7901 cells were fusioned by using of polyethylene glycol,and the pure fusion cells were screened out by cultured with HAT and HT selective culturesystems. The phenotypes of dendritic cells and fusion cells were detected by flow cytometry.Part 2. The biological characteristics of fusion cells (such as their morphologicalspecialities, the expression of cell surface phenotypes (CD80\CD83\CD1a\HLA-DR), theirgrowth curves, whether they can proliferate to be a new carcinoma in nude mice, the abilityto irritate cytotoxic T lymphocytes and anti-tumor immune reactions) were tested to verifytheir safety when be used as anti-tumor vaccines. Part 3. The ability of fusion 3第三军医大学博士学位论文cells-activated T lymphocytes to kill SGC7901 gastric cancer cells was investigated byMTT method in vitro. SGC7901 cells were injected subcutaneously in nude mice to makethe planted gastric cancer models. The anti-tumor effects were evalutated by detecting thegrowth of the new planted cancers, the apoptosis and proliferation of the planted cancercells, the pathologic changes of the tumor tissues and the prohibitive rate for planted tumorspre and after the fusion cell vaccine's application and the cell divide cycles. Part 4. Theexpression of B7-1, B7-2 mRNA of fusion cells were detected by RT-PCR to detect thegene changes of these cells.Results: 1.We obtained mature dendritic cells from gastric cancer patients' peripheralblood mononuclear cells by co-cultured with granulocyte-macrophage colony stimulatingfactors, interleukin-4 and tumor necrosis factor-α. These mature dendritic cells had typicalmorphological characteristics and their phenotypes' expression were CD83 75.54±0.73%,CD1a 64.94±0.52%,CD80 61.56±0.27%,HLA-DR 62.50±0.66%. 2. Dendritic cells andSGC7901 cells could be fusioned by PEG and we could get pure fusion cells by culturedwith HAT\HT selective culture systems. 3. Fusion cells were much larger than dendriticcells or SGC7901 cells. They exhibited typical characteristics by light and electronmicroscope testing. They lived in suspension in the culture medium and could notproliferate to be new carcinomas in nude mice. The cell phenotypes were CD83 80.16±1.12%,CD1a 72.86±2.48%,CD80 81.24±2.76%,HLA-DR 79.54±1.56% respectively.Fusion Cells also could strongly irritate the proliferation of T lymphocytes. 4. Fusionvaccine could induce strongly anti-tumor biologi...