| Tumor is a serious risk to human health. Besides surgery, radiotherapy, chemotherapy and biological therapy methods, more and more attention has been paid to immunotherapy. The purpose of immunotherapy is to control and kill tumor cells through activating immune system, enhancing anti-tumor immunity of the microenvironment. But accumulating evidences showed that tumor cells can escape recognition and attack of human immune system by modifying their own surface antigens or changing tumor microenvironment and thus anergizing immune cell function.All the present studies that investigating detail mechanisms of tumor escape just focusing on changes of tumor cell itself, tumor-associate or -specific antigens or host immune system. Among them, the immune dysfunction of tumor-bearing patients is the main reason for lower level of immune response and more serious tumor escape, and is an important limiting factor for immunotherapy. And the changes of professional antigen-presenting cells (APC), dendritic cell (DC), play an important role for the immune dysfunction. It is through a series of complex processes that immature dendritic cells can be differentiated from bone marrow hematopoietic stem cells and then turned into full-featured mature DC through stimulation. Tumor cells can not only directly induce DC apoptosis, but also affect their number, maturation, function and thus down-regulating their antigen-presenting or immune response-initiating function by interfering with various steps of DC differentiation. It has been proved that at least partial tumor cells can affect DC's function by their membrane molecules or secreted cytokines. However, the detail mechanism is still largely unknown because of the complicated tumor microenvironment and lots of tumor- associated molecules.To investigate the molecules that affect DC's function, three parts of work have been done in the present project. First, obtain some candidate DC-binding molecules from human hepatoma T7 phage cDNA library by four-cycles of bio-panning with cultured immature DCs and then identify specific binding phage clones by ELISA, amplify clones by PCR and do DNA sequencing, homology analysis, bioinformatics analysis to know the progress and function of the molecules. Second, the following experiments are adopted to identify and analyze expression of GDF15 in liver cancer cells and tissues: detect transcription of GDF15 in liver cancer cell lines by RT-PCR (1), protein expression by Western blot (2); detect expression and location of GDF15 in cells by confocal (3) and immunohistochemistry (4). Third, to make sure whether exogenous GDF15 can affect the maturation of co-cultured DCs, we will detect the effect of GDF15 on DC phenotype (flow cytometry), morphology (scanning electron microscope), phagocytosis (flow cytometry) and IL-12 secretion (ELISA). The results of this study are the following:①We have obtained a series of tumor-associated molecules (human ferritin light chain, alpha-fetoprotein,α1-microglobulin precursor, growth differentiation factor 15 etc.) that can specifically bind DCs. And a preliminary assessment of the possible implications, receptors and signaling pathways of these proteins has been done. Among them, the effect of GDF15 on DC has been done in detail.②GDF15 mainly localized in the cytoplasm, was expressed at higher level in the liver carcinoma than normal control.③Maturation of DCs was significantly inhibited in the presence of exogenous GDF15, which indicated GDF15 taken part in the regulation of DC maturation.In summary, we obtained some candidate molecules that may interact with DC through cDNA library phage display method and our preliminary data implicated that one molecule named GDF15 could inhibit the maturation of DC. The present study laid a foundation for further exploring the mechanism of the effect of GDF15 on DC. |
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