IL-15 Gene Therapy of Colon Cancer in Mice
|School||Tianjin Medical University|
|Keywords||Gene therapy Cytokines Interleukin-15 Carcinoembryonic antigen promoter Plasmid vector Electric transfected Tumor|
Biomedical in recent decades has made considerable progress, but the treatment and prognosis of many malignancies remain very optimistic. In recent years, gene therapy has become an important part of the tumor therapy, especially in cytokine-related research, they have been more and more attention. Tumor gene therapy studies have achieved a lot, but it remains to be further studied, including many of the problems of the safety and efficacy of gene therapy vectors. In a previous study, we successfully constructed a dual-promoter IL-15 gene expression plasmid vector pHi2-IL15-CMV-tat (L1), and efficient expression of the IL-15 gene plasmid vector pHi2-turn signal peptide spIL15-CMV-tat (L3), and successfully constructed with CEA specificity of IL-15 expression vector PHI2-IL15-CEA-TAT (L2), of PHI2-spIL15-CEA-TAT (L4). In this study, a plasmid vector constructed above, and another kind of green fluorescent protein express the carrier pHi2-EGFP-CMV-tat (L6) by electrical transduction method, CEA-positive colon cancer cell line SW480 and CEA-negative breast cancer cell lines were transfected MCF-7, using an inverted fluorescence microscope transfection of cells through cell expression of green fluorescent level assessment; transfection efficiency by flow cytometry (FCM) analysis of tumor cells; enzyme-linked immunosorbent assay (ELISA) detection cells transfected on The supernatant concentration of IL-15 in the analysis of the IL-15 gene expression. The results showed that: the use of pHi2-EGFP-CMV-tat (L6) electrically transfected SW480 cells and MCF-7 cells, inverted fluorescence microscope can observe green fluorescent protein expression by flow cytometry analysis of the transfection efficiency of the tumor cells in 10% -30%. Plasmid L1 and L2, L3, L4, and the transfected cells, the supernatant visible expression of IL-15; plasmid PHI2-sp the IL15-CMV-TAT (L3) and the PHI2-spIL15-CEA-TAT (L4) of IL- 15 expression levels higher than pHi2-IL15-CMV-tat (L1) and pHi2-IL15-CEA-tat (L2), the difference was significant (P lt; 0.01); transfected SW480 cells, CEA promoter positively regulated The plasmid L2 is L4 after the start of Positive regulation plasmid L1, L3 of between IL-15 expression level was no significant difference (P GT; 0.05); transfected MCF-7 cells with the corresponding CMV, L1 and L2, L3 and L4 between the IL-15 expression level can be seen a significant difference (P LT; 0.05) plasmid the expression of IL-15 in the L1 is higher than L2, L3 of IL-15 expression levels higher than L4. The in vivo tests showed that intraperitoneal injection of the plasmid carrying the EGFP L6 lower right mice 48hr after in vitro in vivo imager can see the obvious fluorescence peritoneum. Colon tumor cells of mice injected mice source CT-26 the establishment of intra-abdominal tumors in tumor-bearing mice model regularly injection L3, L4 plasmid to observe the impact of ascites and survival of tumor-bearing mice. The results found that injection of plasmid vector L3, L4 higher than the survival time of mice injected with PBS alone and no-load control group. EGFP expression plasmid vector L6 vivo electroporation transfection experiments, mice were injected subcutaneously CT-26 cells to establish subcutaneous tumor model, intratumoral injection of plasmid transfected be electric in vitro in vivo imaging shows the fluorescence reaction, and then take the tumor body tissue for frozen sections, fluorescence microscopy. Visible green fluorescent protein expression was found near the needle tract. Further efficient expression plasmid vector L3 test treatments, the results show that the the a plasmid vector L3 mice tumor growth rate was significantly slower than the control group. The above results show that the electrical transfection method can be effective in vitro and in vivo IL-15 expression plasmid was introduced into the tumor cells, and the same time, the intraperitoneal injection of plasmid can also be the peritoneal effective expression of the target gene. Intraperitoneal injection of IL-15 expression plasmid to some extent inhibited tumor formation and development of peritoneal transplant. Electric transfection can inhibit IL-15 expression plasmid subcutaneously transplanted mouse colon cancer growth. In this study, IL-15-based tumor immune gene therapy clinical application provide an experimental basis.