Experimental Treatment Of Neural Stem Cells Modified By Cytosine Deaminase Gene For Glioma

ObjectiveTo explore the conditions and methods of cell detachment,cultivation, differentiation and identification of the rat embryo neural stem cells in vitro. Transfection, Screening,amplification,identification to obtain stable expression of CD gene-engineered neural stem cells in vitro;To explore the CD gene-modified neural stem cells in vitro and in vivo inhibition the proliferation of glioma to find a new approach for the treatment of glioma.Methods1.The neural stem cells were derived from the cortex and subcortex tissue of rat embryo at the age of 14～16 days and proliferated steadily under the serum-free medium which contained epidermat growth factor(EGF),basic fibroblast growth factor(bFGF) and B27.The neural stem cells clone and passage through limiting dilution assay and differentiatiated in 5% fetal calf serum.The rat neural stem cells and the differentiated neural stem cells were identified respectively by immunofluorescence including Nestin,neuron specific enolase(NSE),glial fibrillary acidic protein(GFAP) and cyclicneucleotide phosphodiesterase(CNP).2.Amplification and identification by restriction enzyme digesting of plasmid pCMVCD In vitro; pCMVCD using liposome-mediated transfection method was transfected into NSCs,G418 screening of positive clones, immunocytochemical identification of recombinants.And detected the efficiency of CD gene and expression of the eukaryotic expression vector.3.The NSCs/CD cells and C6 cells were co-cultured while adding 5-FC, to observe glioma cell growth, Cell survival assay was detected with MTT.Establishment of animal models of C6 gliomas in nude mice,They were randomly divided into test group and control groups.intraperitoneal injection of 5-FC after transplantation of NSCs/CD cells,to observe NSCs/CD-5-FC suicide gene system inhibition of glioma in vivo.Results1.In this experiment,we successfully isolated and identified the 14~16 days pregnant SD rat fetal rat cerebral cortex and cortex of NSCs, and proliferation and differentiation in vitro.2.The pCMVCD plasmid using the liposome-mediated was transfected successfully into NSCs, Could formate the resistant cell clone proliferation after G418 selection.Double-labeled immunofluorescence staining showed that expression of NSCs resistant cell clones of the specific antigen-nestin, and expressed the CD gene at the same time.3.NSCs/CD cells and C6 cells were co-cultured while adding 5-FC,When compared with the control group were significant (P<0.01),Can obviously inhibit tumor cell proliferation.At the same time, significant bystander effects were observed. We successfully established animal model of glioma in nude mice,When transplantion NSCs/CD cells and intraperitoneal injection of 5-FC may be obvious to observe anti-tumor effects. ConclusionCD gene-modified neural stem cells of the suicide gene system inhibited glioma growth,the effect clearly,not only due to the migration ability of neural stem cells,Which can migrate to the surrounding of glioma cells,stable expression of target genes to transformed precursor drugs into the drugs which have the roles of killing tumor cells,thereby inhibiting tumor growth, while the bystander effect is very obvious.The neural stem cells as a purpose gene vector to treat glioma, for the treatment of glioma find a practical feasible path.