Abstract
Objective To construct recombinant adenovirus vectors containing herpes simple virus thymidine kinase (HSV-TK) controlled by human Cfos promoter and cytomegalovirus (CMV) promoter and observe the expression of adenovirus in human glioma U251 cells and their specific cytotoxic effect on U251 cells in combination with ganciclovir (GCV) in vitro. Methods Two recombinant adenovirus vectors containing herpes simplex virus thymidine kinase gene controlled by human Cfos promoter and CMV promoter were constructed (Ad-Cfos-TK-IRES-hr green fluorescent protein [GFP] and Ad-CMV-TK-IRES-hrGFP), respectively. For packaging virus, two adenovirus vectors were transfected into HEK293 cells. Ad-CMV-TK-IRES-hrGFP and Ad-Cfos-TK-IRES-hrGFP were collected, purified and transfected into U251 cells. The expression of EGFP was observed under a fluorescence microscope. Recombinant adenovirus infected U251 cells were cultured with 1, 10, 100 and 1000 μmol/L GCV to observe specific cytotoxic effect on glioma. Results The recombinant plasmid vectors Ad-CMV-TK-IRES-hrGFP and Ad-cfos-TK-IRES-hrGFP were verified by restriction endonuclease digestion digestion and sequencing. Ad-CMV-TK-IRES-hrGFP and Ad-Cfos-TK-IRES-hrGFP adenovirus were collected and purified, successfully. The titers of adenovirus reached 1×1010 IU/ ml. In company with the rising of GCV concentration gradient (1, 10, 100 and 1000 μmol/L), the inhibition rate of U251 cells gradually increased (24.18%±6.01%, 30.39%±9.67%, 57.07%±9.29% and 94.50% ± 3.48%) at mutiplicity of infection (MOI) 100. In company with the rising of GCV concentration gradient (1, 10, 100 and 1000 μmol/L), the inhibition rate of U87 cells gradually increased (9.78% ± 2.24%, 86.33% ± 5.06%, 98.48% ± 0.79% and 98.76% ± 0.93) at MOI 100. Conclusion Adenoviral-mediated delivery of herpes simplex virus thymidine kinase gene controlled by Cfos promoter can confer cytotoxic effect on human glioma cells in vitro. Key words: Glioma; Gene therapy; Adenovirus; Cfos promoter
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