Therapeutic efficacy of VP_(22) and cytosine deaminase gene modified rat neural stem cell in treatment of C6 glioma in vitro

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