| Objective: Cancer is one of the serious disease threatening human health currently. At present, operation, radiotherapy and chemotherapy are the main methods to treat tumors. But because of the limit of stage classification and location of the tumor,operation is restricted. In the course of radiotherapy or chemotherapy, not all of the tumors are sensitive or applicable to these methods. At the same time, long-term therapy of radiotherapy or chemotherapy can also damage immune system, even influence the function of other organs, further induce a new disease. Therefore, to develop some new approach to overcome cancers is necessary. Immunotherapy can not only enhance the immune function of body, but also kill tumors in special pathway, and does not influence the function of other normal tissues. At present, more and more people have pay attention to immunotherapy. Cytokine therapy is one of the focus in immunotherapy research. In previous research, researchers used to injected the protein of cytokine into body with tumor to treat cancer. Because of the short half-life, large usages, strong side-effect, it was limited in clinical application. Therefore, in recent years, researchers have transfer cytokine genes into tumors which have no or less immunogenicity. By this way, some tumor cells had lost the tumorigenicity,and became strong immunogenicity. These modified tumor cells can not only stimulate the immune response of T lymphocytes to kill them, but also to kill unmodified wild tumors specially. To some extent, it exhibits an attractive foreground to treat tumors with cytokine gene therapy.Interleukin 15 is a new cytokine that was found by Grabstein in 1994. It is well known that natural killer cells can kill tumor cells in a broad-spectrum way, CD8+T cells can kill tumor cells in a special way. IL-15 has a critical role in development and function of NK and CD8+ T cells. So it evokes the stong interesting of tumor immunological treatment by using IL-15. IL-15 mRNA is not only widely expressed in heart, lung, kidney, placenta and skeleton muscle, but also expressed in monocytes/macrophages, dendritic cells, fibroblasts,epithelia cells and marrow stroma cells. But researchers found that the expression of IL-15 was regulated by many factors. Long signal peptides of IL-15 is the main disadvantage factor that in the process of secreting IL-15. To substitute IL-2 signal peptide (IL-2sp) for IL-15 signal peptide (IL-15sp) can promote secretion of IL-15. In present study, we substituted with IL-2 signal peptide for IL-15 peptide and constructed a modified IL-15 gene (FIL-2sp-IL-15mp), inserted FIL-2sp-IL-15mp into a eukaryotic expression vector and transfered into tumor cells. Which established a foundation for investigating the tumor therapy with IL-15 gene .Methods: The complete cDNA fragment of hIL-15(FIL-15cDNA) and the DNA fragment encoding hIL-15 mature peptide(FIL-15mp) were amplified by PCR from pCI-IL-15. The DNA fragment of hIL-2 sp was amplified by PCR from pcDNA3-IL-2. Using the method of gene splicing by overlap extension, the fragment of modified IL-15 gene(FIL-2sp-IL-15mp) was acquired. The FIL-2sp-IL-15mp and the eukaryotic expression vector, pEGFP-N1, , were digested with EcoR â… and BamH â… respectively and the FIL-2sp-IL-15mp was inserted into EcoR â… and BamH â… sites of pEGFP-N1 to construct pEGFP-N1/ IL-2sp/IL-15mp (pEGFP-N1/215). By the same means the recombined eukaryotic expression vector pEGFP-N1/IL-15mp ,which contained hIL-15 mature peptide coding sequence, and the recombined eukaryotic expression vector pEGFP-N1/IL-15 , which include FIL-15cDNA were acquired respectively for controls. After identified by PCR, restriction endonuclease and DNA sequencing analysis, the three eukaryotic expression vectors were transfected by the Lipofectamine 2000 into NCI-H446 respectively. These stable transfectsed cells were selected respectively in complete culture medium supplemented with 0.8mg/ml G418 and limited dilute. When the cell lines that tranfected with the e...
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