Study Of IL-24 Gene On The Induction Of Apoptosis In Rat Glioma C6 Cells

Objective:To culture the rat glioma C6 cells in vitro, and the plasmid DNA of IL-24 gene from-E.coli DH5a transfected into C6 cells. To detect the inhibitory effects of IL-24 on the growth of C6 cells in vitro, and explore an effective way to treat gliomas.Methods:To culture the E.coli DH5a with LB medium, and extract the pEGFP-Cl vector and the pEGFP-IL-24 plasmid from E.coli DH5a using plasmid extraction kit. Agarose gel electrophoresis after the vector and the plasmid has been digested with restriction endonuclease Xho I and EcoR I, and measure the concentration and purity of plasmid, in order to check the accuracy of the plasmid DNA. To culture the C6 cells in vitro, and the pEGFP-Cl and the pEGFP-IL-24 were transfected into the C6 cells useing the Liposomes, while the C6 cells as empty-carrier control, and the C6 cells were added Adriamycin as positive control. In order to indicate whether the plasmid was transfected into C6 cells, we observed the expressions of EGFP in the four groups cells by fluorescence microscope. To observe the form of the C6 cells at high microscope after 48h, the survival status of the cells were observed with AO/EB, and the growth of the C6 cells was measured by MTT colorimetric assay in different time. The results were expressed as mean±standard deviation and evaluated with SPSS software by analysis of variance, P<0.05 were consided statistically significant.Results:Restriction enzyme digestion and electrophoresis after extract the pEGFP-Cl vector from E.coli DH5a, the result analysis showed that as expected a 4700bp line has been found. Restriction enzyme digestion and electrophoresis after extract the pEGFP-N1-IL-24 from E.coli DH5a, the result analysis showed that two lines of the plasmid including 4700bp and 660bp line has been found, and it is consistent with the pEGFP-Cl vector size and the size of IL-24 gene. The small line were not find in the plasmid electrophoresis without the restriction. There were high purity in the plasmid according to OD260/OD280 about 1.8. To observe the each group cells under a fluorescence microscope after 24h. We found the C6 cells transfecting pEGFP-Cl and the C6 cells transfecting pEGFP-IL-24 emitted green fluorescence, but the cells of without transfecting plasmid group didn't. The result showing that the plasmid DNA can be transfected into C6 cells and expression in cells. To observe the form of the four groups cells at high microscope after 48h. We can see, the C6 cells which transfecting. pEGFP-IL-24 and adding Adriamycin were shrinkage and they didn't attach to the bottle, however, the C6 cells and the cells transfecting pEGFP-Cl were attach to the bottle. The C6 cells showed spindle or polygon. AO/EB fluorescence staining of the four groups cells showed that most of the C6 cells which transfecting pEGFP-IL-24 and adding Adriamycin were stained orange-yellow or red. Part of the cells were apoptotic cells, and they showed cracking. Most of the C6 cells and the cells transfecting pEGFP-Cl were stained green, and the cells large and round. The MTT colorimetric assay showed the C6 cells of the growth didn't had significant difference between the C6 cells transfecting pEGFP-IL-24 and the C6 cells adding Adriamycin (P>0.05). But the C6 cells of the growth had significant difference between the C6 cells transfecting pEGFP-IL-24 and the C6 cells transfecting pEGFP-Cl (P<0.05), the same between the C6 cells transfecting pEGFP,IL-24 and the C6 cells (P<0.05). The growth rate of the C6 cells transfecting pEGFP-IL-24 and adding Adriamycin was inhibited, while the cell growth curve of empty-cartier group was similar to the C6 cells transfecting pEGFP-C 1.Conclusion:1. The pEGFP-Cl vector and the pEGFP-IL-24 plasmid has been extracted successfully, and the gene sequence of the vector and plasmid is correct through restriction enzyme digestion and electrophoresis.2. The pEGFP-Cl and the pEGFP-IL-24 can be transfected into C6 cells using liposome method, and they can be expressed in C6 cells.3. The IL-24 gene can induce apoptosis of glioma C6 cells, and inhibit proliferation of C6 cells in vitro.4. The IL-24 gene for the glioma gene therapy may have lurking value.