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Objective: To construct containing the target gene IL-24 eukaryotic expression plasmid pEGFP-N1-IL-24, its transfected mouse melanoma B16 cells to study intracellular IL-24 expression on tumor cell apoptosis and proliferation in vitro the impact. Methods: pUC57-IL-24 with the restriction endonuclease after double digestion enzymes, was recovered by agarose gel electrophoresis, purified, connected with the pEGFP-N1, the ligation product was transformed into competent cells E.coli DH5a in plasmids were extracted and digested electrophoresis again to check the accuracy of the IL-24 inserted into the pEGFP-N1 and sequencing verified. Liposomes pEGFP-N1-IL-24 transfected B16 cells (B16/pEGFP-N1-IL-24 group), the set of untreated B16 cells (B16 group) for the control group, the other only added fat The plastid B16 the cells (B16/Lip group) for the liposome group, pEGFP-N1 transfected B16 cells (B16/pEGFP-N1 group) for the empty vector, B16 the cells (B16/VCR group join vincristine ) for chemotherapy drugs group observed the expression of the cells in each group after 24 hours under a fluorescence microscope, inverted microscope high magnification observation cell morphological changes at 48h after transfection with acridine orange / ethidium bromide (AO / EB ) method to detect cell apoptosis was detected by MTT B16 cell proliferative capacity in vitro experimental results expressed as x ± s, using statistical software to do a t-test, P lt; 0.05, significant difference. Results: 1. Construct containing IL-24 eukaryotic expression plasmid by restriction enzyme digestion, electrophoresis, discovered the bands of 4.7kb and 660bp, in line with the expected value, verified by sequencing pEGFP-N1-IL-24 gene sequence . The B16, B16/Lip B16/pEGFP-N1 B16/pEGFP-N1-IL-24, B16/VCR five groups of cells respectively transfected 24h observation, visible under a fluorescence microscope B16/pEGFP-N1 group the cells and B16/pEGFP-N1-IL-24 cells emit green fluorescence of EGFP gene expression in B16 cells, while the other group of cells no green fluorescence. 3. Observe of of B16, B16/Lip, B16/pEGFP-N1 B16/pEGFP-N1-IL-24 group B16/VCR five cells for 48h after transfection cell morphology, visible, B16, B16/Lip group and The full vision B16/pEGFP-N1 group of cells, cell proliferation, cell similar in size, circular, oval or short shuttle-type, clear extracellular week, three-dimensional. B16/pEGFP-N1-IL-24 group and B16/VCR group of cells, most of the cells surrounding the more obscure, tightly clustered into a group, a small number of cells, fragments of irregular size. 4. B16 B16/Lip, B16/pEGFP-N1 B16/pEGFP-N1-IL-24, B16/VCR five sets of cells were AO / EB double staining, the results show that the B16 group B16/Lip group and B16 / pEGFP-N1 cells mostly as the nuclear chromatin green, and was normal structure, a small amount of dead cells, nuclear chromatin with red and showed normal structure; B16/pEGFP-N1-IL-24 group, and B16/VCR of group cells most of the apoptotic cells, the nuclear chromatin orange or orange-red, and was pyknotic-like or bead-like. 5. Of B16, B16/Lip, B16/pEGFP-N1 B16/pEGFP-N1-IL-24, B16/VCR five groups of cells with MTT colorimetric assay B16/pEGFP-N1-IL-24 group the B16/Lip group, B16/pEGFP-N1-IL-24 with B16/pEGFP-N1 inhibition rate there is a significant difference between (P lt; 0.05, P lt; 0.05) and B16/pEGFP-N1-IL-24 groups with B16/VCR inhibition rate of no significant difference (P = 0.991). By 48h after transfection, B16 cells done by the inhibition rate histogram can be seen B16/pEGFP-N1-IL-24 group, and B16/VCR of cells the proliferation and B16/Lip group and B16/pEGFP-N1 group significantly suppressed. Conclusion: 1. Successfully constructed recombinant plasmid pEGFP-N1-IL-24, and to verify the correctness of the gene sequence by restriction enzyme digestion, electrophoresis and gene sequencing. Exogenous IL-24 gene in vitro transfection of mouse melanoma B16 cells, and expression in B16 cells. Exogenous IL-24 gene can induce apoptosis in the mouse melanoma B16 cells, inhibit their proliferation in vitro. 4.IL-24 gene as a melanoma therapeutic genes has broad application prospects.
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