A preliminary study on the construction of double suicide gene delivery vectors by mesenchymal stem cells and the in vitro inhibitory effects on SKOV3 cells

The aim of the present study was to investigate the efficacy of using human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) as gene delivery vectors in the treatment of ovarian cancer. Lentivectors overexpressing cytosine deaminase (CD) and herpes simplex virus thymidine kinase (HSV-tk) (pGC-FU-CD-TK) were constructed, and confirmed by enzyme digestion, DNA sequence and western blotting. Quantitative PCR (PCR) was used to verify the overexpression of the fusion gene (CD and HSV-tk). SKOV3 cells were co-cultured with MSCs/tk+CD+ at a 1:1 ratio, and were then treated with the prodrugs (GCV) and/or 5-fluorocytosine (5-FC) at different concentrations, and the cytotoxic effects were evaluated using MTT assay and flow cytometry. DNA sequencing demonstrated that the sequence of HSV-tk and CD genes were consistent with the objective sequence and western blotting verified that the constructed lentivector could produce the HSV-tk/CD gene. The packed titer was 2.00e+8 TU/ml. The pGC-FU-CD-TK could be stably transferred to hUCB-MSCs, and the infection efficiency was almost 80%. RT-PCR demonstrated that the expression levels of the HSV-tk/CD fusion gene in MSCs/tk+CD+ group was 75 times that in the negative control (P