Objective: To observe the cell uptake kinetics and specific gene expression effect of hTERT antisense molecular probe in vitro.
Methods: Antisense molecular probes targeting hTERT mRNA were radiolabeled with technetium-99m by the method of bifunctional chelator. HepG2 cells expressing hTERT were cultured. The uptake kinetics of molecular probes mediated by liposome or not in cells were examined in vitro. RT-PCR (reverse transcriptase polymerase chain reaction) method was performed to assay the specific gene expression effect of the molecular probes. All data were analyzed by the statistic software of SPSS 12.0.
Results: The labeling efficiencies of molecular probe reached (76 +/- 5) % (n = 5), the specific activity was up to 1.850 x 10(6) Bq/microg, and the radiochemical purity was above 96% after purification. The absolute accumulation of (99m)Tc, whether on antisense or sense molecular probe, was clearly higher with liposome-mediated than without liposome-mediated (P < 0.05). Furthermore, liposome advanced the peak time of cellular uptake of antisense molecular probe, with the highest accumulation occurring at the end of 2 h, and remaining at the same level till 3 h later. In comparison with sense molecular probe, antisense molecular probe preserved the capacity to bind living hTERT-expressing cells specifically and inhibited the expression of hTERT mRNA significantly as well as ASON.
Conclusion: Liposome-mediated method could increase cell uptake of molecular probes in vitro. Antisense molecular probe preserved the capacity to inhibit the expression of targeting mRNA specifically. All results provided the basis for further in vivo study.