ODD调节HSV-TK／GCV对乏氧环境宫颈癌Hela细胞特异杀伤作用的研究

0bjective To observe the specific killing effects of a eukaryotic expression vector expressing herpes simplex virus thymidine kinase regulated by ODDD(oxygen dependent degradation domain)on Hela cell under hypoxia which makes it possible to further investigate their potency of antitumor potency under hypoxia. Methods The encoding sequence for ODDD amplified with RT-PCR and inserted into pEGFP-C1;The HSV—TK was amplified by PCR and cloned into the multiple clone site of pEGFP-C1 and pEGFP-ODDD to obtain pEGFP-TK and pEGFP-ODDD-TK recombinant plasmid.Transient transfected cells with pEGFP-ODDD were exposed to hypoxia 24 h after transfection. Relative EGFP fluorescence was measured by a flow cytometer under hypoxia and normoxia . The sensitivity of Hela cell line transfected with pEGFP-ODDD-TK and pEGFP-TK recombinant plasmid to the drug GCV was determined with the method of MTT after treating with GCV for five days．Results The integrity reporter vectors were verified by restriction enzyme digestion. the EGFP expression levels of pEGFP-ODDD in normoxia in Hela cells were 11.96％ and were lower than those of hypoxia sample39.92％. Comparison of the sensitivity of cells to GCV showed that the cells in pEGFP-ODDD-TK groups under hypoxia were much more sensitive to GCV than the cells under normoxic condition(P<0.01)，the higher the GCV concentration，the greater the difference .Conclusion ODDD could ruduce expression of EGFP in Hela cells under normoxic condition. ODDD could specify the kiling effect of HSV-TK ／GCV system on Hela cell line under hypoxic condition.