Bone Marrow Mesenchymal Stem Cells Transfected With Cytosine Deaminase Gene And Brain Glioma Therapy

The glioma, a common kind of primary brain tumor, accounting for more than 50% of intracranial primary tumor, is difficult to be treated so far. Cytosine deamimase (CD) and Bone marrow mesenchymal stem cells (BMSCs) are genes for glioma therapy and cell carrier which have been widely concerned, the CD gene is carried to somewhere close to the glioma cells by BMSCs, and was managed to play an important role of chemotherapy prodrug for conversion and effective gene therapy around glioma cells.In the present thesis, pIRES2-AcGFP1-CD eukaryotic expression vector was initially constructed, and then CD gene was transfected into rabbit and mouse bone marrow mesenchymal stem cells, respectively, through liposome mediated method, the cytosine deaminase gene was also successfully cloned, and the result of gene sequencing was consistent with that published in Genbank. The cytosine deaminase genes were subcloned into pIRES2-AcGFP1 plasmid, and pIRES2-AcGFP1-CD eukaryotic expression vector was thus constructed. The results indicated that pIRES2-AcGFP1-CD eukaryotic expression vector had been successfully transferred into bone marrow mesenchymal stem cells.In addition to the above described liposome-mediated method, CD gene packaged by lentiviral vector was also attempted to transfect BMSCs. The ability of transfection and expression of the CD lentiviral vector constructed could meet the demands of in vitro and in vivo experiments, respectively. Moreover, the BMSCs-CD/eGFP was constructed successfully, and was subsequently verified by gene sequencing and PCR testing. The transfection efficiency of BMSCs transfected by lentiviral vector-mediated CD gene was much higher than that of transfected by liposome carrier, therefore, in the subsequent experiments, the mouse BMSCs transfected by lentiviral vector-mediated CD gene that were selected.After the mouse BMSCs with stable expression of CD gene were successfully obtained, the transfected BMSCs with CD gene were cocultured with glioma cells with 5-FC adding into this culture system, and then the apoptosis of glioma cells was detected through flow cytometry. The results confirmed that the transfected mesenchymal stem cells could effectively transform chemotherapy prodrug 5-FC into chemotherapy drug 5-FU with cytotoxicity, and was able to inhibit the growth of glioma cells in vitro. Subsequently, the C6 glioma model was in situ inoculated with previous transfected BMSCs carrying CD gene. Following to this, the 5-FC was intraperitoneally injected and its inhibitory effect on glioma cell growth was also observed. In order to approach the clinical treatment process, the treatment should be started after the tumor is being formed. The relative symptoms emerged after 7 to 10 days of cell inoculation, and the tumor formation can also be seen by MR, so the 7th day was chosen as the starting time of treatment. The results showed that the BMSCs-CD/eGFP could significantly improve rats’ symptoms of increased intracranial pressure and prolong their survival time. Meanwhile, the tumor volume from treatment group was obviously smaller than that of the control group; tumor necrosis, hemorrhage and other positive changes were able to be observed through HE staining in treatment group but were relatively seldom in the control group. At the same time, BMSCs were labeled by tracer eGFP, and their distribution was further observed in tumor tissues, the results confirmed that the BMSCs-CD/eGFP cells distributed in the interior and edge of C6 glioma tissues, and mainly distributed on the irregular edge of glioma, showing wrapping-like shape.Molecular biological techniques were combined with morphology assays in this study, CD gene was transfected into rat BMSCs by lentiviral vector carrying enhanced green fluorescent protein (eGFP) by transgenic technology, and the glioma therapy through transfected BMSCs with CD gene in vivo and in vitro was further investigated and observed. The results showed that the BMSCs could migrate to glioma cells, and the transfected BMSCs with CD gene could also inhibit the growth of glioma cells in vivo and in vitro and thus to prolong rats’survival time.