| Human chorionic gonadotrophin (hCG), a glycoprotein hormone secreted by fetal syncytiotrophoblast in pregnant woman, plays an important role in maintaining the function of lutein and inhibiting fetal rejection by mother. Much attention had been paid to the relation between ehCG and tumor since Braunstein and his colleagues reported that ectopic human chorionic gonadotrophin was expressed in syncytiotrophoblast tumor cells and non-syncytiotrophoblast tumor cells. According to our previous research and others' studies, ehCG was expressed in many kinds of tumors, it seems that the expression of ehCG in tumor cells and tissues was ubiquitous as long as the cells differentiate into tumor cells.It is intriguing that the hCGβ??dimer secreted by tumor performs the function of growth factor that enhances and regulates the proliferation, differentiation and growth of tumor cells, so that the tumor cells can grow in microenvironment autonomously. Moreover, the expression of membrane-associated ehCG, containing six oligosaccharide side chains with negative charge, made the tumor cells susceptible to sialyation, which may relate to the immune anerge or immune tolerance. Therefore, ehCG can be used as an ideal target tumor antigen to develop a novel tumor vaccine for clinic immunotherapy for treatment of tumor.Genetic immunization has developed as a new immunological technology since 1990s. Its rationale is that the eukaryotic expression vector containing a target antigen-coding gene is directly injected into the tissues to express a protein that induces specific immune response. Genetic immunization not only induces specific humoral immune response but also effectively induces specific cellular immune response. At the same time, the immune stimulatory sequences (ISS) containing CpG motifs in vector DNA performs the immune stimulatory function as an important and non-specific adjuvant. Therefore, genetic immunization can be potentially a powerful immunotherapy for treatment of tumor.As mentioned above, we here introduce a novel immunological technique-gene immunization, and select ehCG as a target antigen to study the immune response and its anti-tumor effects.PART I: Study on the Relationship between ehCG and Tumor To investigate the relationship between the expression of ectopic human chorionic gonadotrophin (hCG) and the development of malignant tumor and determine the feasibility of ectopic hCG as a target antigen of anti-tumor vaccine, sixty serum samples from tumor patients, twenty-three tissue samples of intestinal adenocarcinoma and ten tumor cells lines were collected to investigate the expression of ehCG using radioimmunological assay and immunocytochemical technique as well as flow cytometry technique. The results showed that seventeen of sixty cases of serum samples and seven of twenty-three tissue samples of intestinal adenocarcinoma were positive in ehCG. The expression of membrane-associated ehCG was tested in most of the tumor cells irrespective of their types or origins. While observing and analyzing the stained tissues, we found that the ehCG was expressed in the poorly differentiated cells, which were located at the frontier of metastatic invasion. These results suggested that ehCG expressing in the secretory pool or membrane-associated pool was associated with histological differentiated degree of carcinomas, the development of malignant tumor and the metastatic potential of cancer cells. Therefore, ehCG may be a candidate as a target tumor antigen for active immunotherapy.PART II: Construction of a Novel Tumor Gene Vaccine Based on ehCGβ and its Function in Inducing Specific Immune Response 1. Construction of a Novel Tumor Gene Vaccine based on ehCGβ ehCGβ encoding gene, amplified by PCR and modified by restriction endonucleases digestion with BglII and BamHI, was subcloned into an eukaryotic expression vector TR421 to construct a recombinant plasmid TR421-ehCGβ that was identified by PCR or restriction endonucleases digestion. DNA sequencing showed that the... |
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