| Natural killer (NK) cells play an important role in innate immune responses against transformed and infected cells. They can efficiently kill the target cells without a need for prior sensitization and wouldn't damage the normal cells. NK cells mainly appears in the spleen, bone marrow, lymph nodes and peripheral blood, they would move to inflammation sites when induced by various chemoattractants. There are many inhibitory and activating receptors on the cell surface, through which signals can be transduced to regulate the activities of NK cells. NK-92 cells is a kind of human NK cell lines, which was isolated and established from a patient with non-Hodgkin's lymphoma, can be easily cultured and expanded in vitro and was widely used for therapeutical researchs.NKG2D is a multi-subunit cell surface receptor expressed byαβCD8 T cells,γδT cells and NK cells, belongs to the c-type lectin-like receptor family. It is an important activating receptor of NK cells and interacts with various ligands. Major Histocompatibility Complex class I related Chain A (MICA) is a ligand for receptor NKG2D, which consists of 383 amino acids and can be devided into three parts: three extracellular domains ( 1,2 and 3), a transmembrane region and a cytoplasmic tail. Under normal conditions, MICA is only can be found on fibroblasts, gastrointestinal epithelium and endothelial cells. However, it is up-regulated on many tumor cells which are more sensitive to NK cells.Herceptin is a recombinant humanised monoclonal antibody which was approved by US FDA for treatment of Her2-overexpression breast cancer. It can target the extracellular domain of the Her-2 protein and inhibit the growth of tumors. Despite the success of Herceptin when in combination with chemotherapy, the overall survival is low and much works are still need to do.To obtain a better anti-tumor activity, we think about to construct a fusion protein of Herceptin with MICA which can bind to a Her-2 positive tumor cell and an NK cell. In this way, not only the anti-tumor activity of NK cells can be enhanced by MICA, but also the number of NK cells attracted by tumor cells will increase. As a result, tumors would be killed more efficiently.We constructed a fusion gene composed of Herceptin and the extracellular domain of MICA through Overlap-PCR. Then used a adenovirus carrying the fusion gene to infect 293 cells to produce the fusion protein Herceptin-MICA(E). Enzyme-linked immunosorbent assay (ELISA) and Western blot showed that the recombinant adenovirus could normally express the fusion protein and the concentration in suspension arrived at (471.93±12.33)ng/ml after 48-hour infection.In Indirect immunofluorescence Assay (IFA) ,fluorescence was observed that in the SK-OV-3 cells which were treated with the fusion protein and the intensity was similar to the commercialized Herceptin. According to Cytotoxicity assay (LDH release analysis) we found that the anti-tuomor of NK-92 cells against SK-OV-3 cells ,which were preincubated with Herceptin-MICA(E), was significantly improved at all different E:T ratios(1:1,3:1,10:1 and 30:1) compared with the control group and the cells that were treated with Herceptin. No apparently differences were observed between the control group and the Herceptin preincubated group.
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