| Colon carcinoma is one of the most common malignancies and also one of the most difficult tumors to cure because of its distant metastasis and easy recurrence, with the liver being overwhelmingly the most common site of metastasis. For patients who are not candidates for surgical resection, prognosis and survival vary with extent of the disease and performance status. The overall median survival is approximately 12 months. Because effective therapy is unaviable, we propose to study the use of gene therapy as a novel treatment for this disorder. The nature of metastatic colorectal cancer makes it well suited for in vivo gene therapy. Metastases are often isolated to the liver and easy accessible to a regional gene therapy strategy. Among immunomodulatory cytokines, interferon- Y (IFN- Y ) induces strong antitumor immunity via several distinct mechanisms. IFN-Y is able to activate a variety of host immune cells, such as T lymphocytes, natural killer (NK) cells and macrophages. IFN-Y is also a potent inducer of MHC class I and II antigens and other cell surface antigens, which increases the antigen-presenting capacity of cells. Many investigators have reported the significant growth inhibition of tumor cells by IFN- Y - In this experiment, we present a strategy for the colorectal cancer treated by IFN-Y gene therapy to investigate the possible anti-tumor effect and its mechanism.1.Construction of IFN- Y gene expression vector, Gene transfection and expression.IFN- Y has been described as an immune interferon. The gene is located on human chromosome 12, encoded by a single gene of 6kbwhich contains four exons and three introns, that gives rise to a 166 amino acid polypeptide containing a 23 amino acid signal sequence. c-DNA of IFN- Y is subcloned into pBluescript after BamHI and EcoRI digestion from pLXSN-IFN-Y , then the IFN-Y cDNA was inserted into pcDNAS expression vector digested by Hind III and Xbal from pBluescript-IFN- Y -pcDNA3-IFN-Y was transfected into LOVO, SW620, HCT116BG cell lines by Lipofectamine. The integration and expression of IFN-Y were identified by PCR and RT-PCR respectively, it showed that the integration and expression of IFN-Y were successful. The activity of IFN-Y expressed in different cell lines was measured by ELISA, which showed that the IFN-Y can be detected 4 hours after gene transfer. Twenty-four hours after gene transfer, the IFN- Y 's activity get the max and maintain for at least 6 days without obviously decline, the IFN- Y expression amount varied in the different cell lines for the diversity of biology between the cell lines. 2.The anti-tumor effects after gene transfer in vitro.We analyzed the expression of cell surface antigen, the growth inhibition of tumor cell and the impact of cell period to explore the tumor cell responses after IFN- Y gene transfer. The supernatants from each cell lines after 48 hours of gene transfer were collected to measure carcinoembryonic antigen (CEA) concentration using radio-immunity test. The results showed that the amount of CEA increased obviously (p<0.01) in LOVO, HCT116BG cell lines which are high expression of CEA originally. However, there is no detectable increase in the supernatants of Hela, SW620 cell lines which originally have little expression of CEA. From the results of flow cytometry analyze, the HLA-DR expression in each cell lines increased significantly(p<0.01) after IFN-Y gene transfer, and the IFN-Y gene transfer can effectively induce theapoptosis of tumor cells, the proportion of DMA synthesis period deceased gradually after IFN- Y gene transfer. We can infer from that the synthesize of DMA be repressed and the proliferation of tumor cells be inhibited. 3. The anti-tumor effect after gene transfer in vivoWe investigated the anti-tumor effect of IFN-y gene transfer treatment in a pre-established murine model with CT26 colon carcinoma. CT26 is a murine colon carcinoma cell line derived from BALB/c mice. BALB/c mice were inoculated subcutaneously(s.c.) or intra-abdominal cavity with CT26 cel... |
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