| Background: Breast cancer is one of the most common malignant tumors in clinical practice. With the conventional therapeutic methods, such as operation, radiotherapy and chemotherapy, the patients acquired unfavourable prognosis. The emerging of gene therapy may solve this formidable problem. In recent years, researchers are interested in the dendritic cell that can present antigens to immunologic cells and induce effective anti-tumor response in vivo. Owing to the lack of specific tumor antigens, the dendritic cells (DCs) cannot be activated to acquire special anti-tumor immune response with the traditional methods. However, the survivin gene was found being highly expressed in most malignant tumors, especially in breast tumors, and it has never emerged in normal cells. Based on these research results, DCs vaccine modified by human survivin gene was presumed to have good effect on treating breast tumors. Thus, in this study, DCs vaccine modified by survivin was constructed with the gene transfection technologies, and the biology activity of this kind of vaccine was studied.Methods: 1. The full-length human survivin cDNA was amplified from recombinant plasmid pCITE2a+/survivin by PCR and the product was inserted into pAdTrack-CMV to construct pAdTrack-survivin according to the gene homologous recombinant principle. The competent E.Coli BJ5183 was co-transfected with shuttle plasmid pAdTrack-survivin and adenovirus skeleton plasmid pAdEasy-1 to form recombinant adenoviral vectors Ad-survivin. The recombinant adenovirus vector was identified by enzyme digestion, and then the recombined virus was amplified in the HEK293 cells to obtain high titer recombinant virus. 2. The mature DCs derived from umbilical cord blood mononuclear cells being induced by rhGM-CSF, rhIL-4 and rhTNF-a were obtained. They were identified through observing the morphological characteristics by inverted microscope and electron microscope, and their phenotype was analyzed by flow cytometry with a few antibodies, such as CDla, CD83, CD80 and HLA-DR. The DCs vaccine with survivin was identified by RT-PCR and Western Blot. 3. The proliferation ability of recombinant DCs vaccine oncytotoxic T lymphocytes was assayed with the 3H-TdR incorporation method, and the concentration of y-IFN in culture supernatant was detected by ELISA method. At the same time, the biological function of DCs vaccine in vitro was analyzed by MTT method. The apoptosis of breast cancer transplantation tumor cells in nude mice was observed by flow cytometry. The effect of the vaccine on the pathological form of the tumor tissue was observed by light microscope.Results: 1. The recombinant adenovirus vector with human survivin gene was constructed successfully. 2. The mature DCs were obtained from umbilical cord blood mononuclear cells that were induced by rhGM-CSF, rhIL-4 and rhTNF-a. The mature DCs had typical morphological characteristics and expressed a few apparent differential cytokines, such as: CDla 46.2±4.3%. CD80 64.2±2.7%> CD83 80.5±2.2%, HLA-DR 84.3±3.8%. 3. The modified DCs vaccine improved the proliferation of T lymphocytes and accelerated the secretion of y-IFN of T lymphocytes markedly in vitro when compared with that of controls. The apoptosis rate of tumor cells treated with the vaccine (54.9411.53%) increased significantly when compared with the control groups (20.94±0.53%, p<0.01).Conclusions: The mononuclear cells that were isolated from human umbilical cord blood were induced into mature DCs successfully by rhGM-CSF, rhIL-4 and rhTNF-a. The mature DCs had typical morphological characteristics and expressed a few differential cytokines. The recombinant survivin modified DCs vaccine induced more intensive anti-tumor effects by irritating the proliferation of T lymphocytes than that of the regular DCs. Otherwise: the recombinant gene vaccine inhibited the tumor growth and induced the apoptosis of breast tumor cells in vitro and in vivo.
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