Abstract
Objective To explore the therapeutic efficacy of rat neural stem cells genetically engineered by VP_(22) and cytosine deaminase (CD) gene in the treatment of C6 glioma cells in vitro. Method The encoding CD gene fragment was got from pAdTrack-CMV-CD by polymerase chain reaction (PCR),and cloned into pHIV-EGFP and pHIV-VP_(22)-EGFP to yield pHIV-CD-EGFP and pHIV-VP_(22)-CD-EGFP, the constructors were confirmed by the restriction enzyme method and PCR. The three-plasmid system containing constructors of pHIV-EGFP plasmid, helper plasmid and envelope plasmid were co-transfected into 293T cells. The viral particles were collected, and infected rat neural stem cells(NSCs) which were cultured from pregnant SD rat brain. The expression of CD transcripts in NSCs/CD -EGFP and NSCs/VP_(22)-CD-EGFP were identified by RT-PCR. The NSCs transfected by lentivirus-EGFP, lcntivirus-CD-EGFP, lentivirus-VP_(22)-EGFP or lentivirus-VP_(22)-CD-EGFP were co-cultured with C6 glioma cells at a ratio of 1:1 respectively. 5-FC was added to the mixed cell cultures for 3days at the final concentration of 100 mg/L and cell viability was performed by MTT assay. Results The cloning sites of the encoding CD fragment in pHIV-EGFP and pHIV-VP_(22)-EGFP were confirmed by the restriction enzyme method and PCR. In the presence of 5-FC, the cell survival rates of C6 glioma cells co-cultrued with the rat NSCs transfected by lentivirus-EGFP, lentivirus-CD-EGFP, lentivirus-VP_(22)-EGFP or lentivirus-VP_(22)-CD-EGFP were (95.57±4.83)%, (49.96±7.19)%, (94.25±4.32)% and (28.06±6.26)% respectively, showed significant growth inhibition (P<0.01) in the presence of CD gene,and the presence of VP_(22)-CD gene were much more sensitive to 5-FC than the presence of CD gene only(P<0.01). Conclusions VP_(22) increases the cell-killing effect of neural stem cells modified by CD gene in C6 glioma in vitro. Key words: Lentivirus vector; VP_(22) shuttle protein; Neural stem cells; C6 glioma cell
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