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Osteosarcoma (osteosarcoma) is the most common primary bone malignancies in children and adolescents. 1970s, the main treatment is amputation and joint amputation, surgical treatment to appear the lung metastases average time for 8 months, 5-year survival lt; 25%. With the development of surgical techniques and imaging, neoadjuvant chemotherapy and immunotherapy progress osteosarcoma cure rate has been significantly improved, and the 5-year survival rate of 80%. But current treatment still unable to solve metastasis, recurrence and drug resistance of tumor patients, tumor gene targeting molecular therapy appear to open up a new therapeutic approach for the treatment of osteosarcoma. Bid (BH3-interacting domain death agonist), containing only the BH3 domains of Bcl-2 family member protein, the full length of 195aa, a molecular weight of 22kDa. The Bid in the cytoplasm, Caspase-8 gathered activated, can cause death receptor-mediated apoptosis signaling pathways, activation of Caspase-8 Cut Bid removal of the N-terminal fragment of the formation of the active truncated Bid ( truncated Bid, tBid) molecules. tBid molecular weight of 15kDa, translocation from the cytoplasm to the mitochondria and other mitochondrial apoptotic molecules regulate apoptosis process by regulating the release of cytochrome c (Cyt c). The oncogene erbB2 encoded product HER2/neu molecules encoding gene is located on human chromosome 17q21, is a transmembrane protein with a protein tyrosine kinase activity, is one of the epidermal growth factor (EGF) receptor family members. Under normal circumstances, HER2/neu only expressed in the fetal period to adulthood, only low levels of expression on the cell surface. HER2/neu overexpression exist in a variety of tumors, such as metastatic breast cancer (25% -30%), inflammatory breast cancer (50%), metastatic ductal carcinoma (60-70%), non-small cell lung cancer (13-55%), ovarian cancer (18-43%), endometrial cancer (10-52%), colorectal cancer (33-85%), kidney cancer (22-36%), head and neck cancer (16 -50%), gastric cancer (21-64%), esophageal (10-26%), prostate cancer (5-46%). Another 40-44% into osteosarcoma and 58% into osteosarcoma lung metastases become an accepted tumor markers. This study will be the signal peptide, a single chain antibody, having the role of membrane translocation of the peptides derived from Pseudomonas aeruginosa and tBid fusion construct secretory targeting pro-apoptotic molecules Immuno-tBid (pCMV-e23sFv-PE Ⅱ (253-364 )-tBid61, namely pCMV-5-2), so that it specifically recognizes, internalization and translocation into the cytosol and induce apoptosis of HER2-positive tumor cells. First liposomal transfection method tBid gene transfected osteosarcoma cell line SOSP-9607, by indirect immunofluorescence, flow cytometry, and electron microscopy methods, the pro-apoptosis activity of tBid detection. Experimental results show that tBid in osteosarcoma cells can be effectively expressed pcDNA3 empty vector transfected control cells with normal morphology, cell membrane integrity, while the experimental group transfection pcDAN3-tBid61 cells become less morphological changes, volume becomes small and pycnosis. Transfected cells the electron microscope and found that the cell membrane to the cell nucleus concentrated chromatin aggregation characteristics of apoptosis. Using flow cytometry, Annexin V and PI double staining, monitoring cell surface phosphatidylserine valgus situation, with cells in the control group (6%) compared to the experimental group early tune death and apoptosis secondary mortality 35.8%. The active tBid further liposomal transfection of the recombinant fusion protein gene (pCMV-5-2) transfected with HER2-positive osteosarcoma SOSP-9607 cells were observed on osteosarcoma SOSP-9607 cells apoptosis induced. By indirect immunofluorescence method to detect apoptotic morphological changes observed in cells transfected target gene pyknosis, nuclear enrichment characteristics of apoptosis. Promote further by Annexin V staining, flow cytometry, and the control group cells (6%) compared to the apoptotic rate was 42%, indicating that the surface of the cell membrane phosphatidylserine valgus description of the gene expression of recombinant Immuno-tBid apoptosis. PCMV-5-2 gene encapsulated liposomes, intramuscular injection of nude mice bearing HER2-positive osteosarcoma (SOSP-9607) can be observed in the tumor growth rate is slower than the control group to the treatment group. Immunohistochemistry results showed that the Immuno-tBid protein selectively distributed in the tumor tissue and muscle tissue of nude mice in the treatment group, and other organizations, such as the heart, liver, spleen, lung, kidney, as well as in the control group tumor staining negative or weakly positive, which may be the endogenous Bid expression of results. Further TUNEL staining of tumor tissue, can be detected in the treatment group, tumor cells showed TUNEL-positive. The above results show that the Immuno-tBid selectively distributed in HER2-positive tumor tissue, and effectively kill cancer by causing tumor cell apoptosis. In summary, the Immuno-tBid gene into the cells of the body can be targeted anti-HER2-positive osteosarcoma cells, is expected to provide a new way of thinking for HER2-positive osteosarcoma treatment.
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