四环素调控HSV-tk基因在HeLa细胞内表达及抗肿瘤作用

Tetracycline regulated HSV-tk gene expression and anti-tumor effect in HeLa cells

目的探讨应用四环素调控单纯疱疹病毒胸苷激酶(HSV-tk)基因在HeLa细胞内表达及抗肿瘤作用。方法将含有四环素阻遏蛋白基因的质粒载体pcDNA6/TR与含有四环素反应元件及HSV-tk基因的质粒载体pcDNA3.1-TetO2-TKI先后稳定转染HeLa细胞,给予不同浓度的四环素类似物强力霉素,应用RT-PCR检测细胞内HSV-tk基因表达,应用MTT法测定转染HSV-tk基因的HeLa细胞对不同浓度无氧鸟苷(GCV)杀伤的敏感性。结果转染两种质粒载体的HeLa细胞经不同浓度的强力霉素作用48h后,RT-PCR结果显示,随着强力霉素浓度的增大,HSV-tk基因表达量逐渐增加,强力霉素4μg/ml时,基因表达的水平达到高峰;加入强力霉素后,该细胞对GCV杀伤细胞的敏感性增加。结论本研究成功地应用四环素基因表达调控系统调控了HSV-tk基因在HeLa细胞内表达及杀伤肿瘤功能,为进一步探讨自杀基因杀伤肿瘤的可调控性提供理论基础。

Objective To investigate the effect of tetracycline regulated HSV-tk gene expression and anti-tumor in HeLa cells.Methods The plasmid vector pcDNA6/TR containing the tetracycline repressor protein gene and pcDNA3.1-TetO2-TKI containing tetracycline response element and HSV-tk gene both were stably transfected into HeLa cells.After treated with different concentrations of tetracycline analogue doxycycline,expression of HSV-tk gene was detected by RT-PCR ,determination of the sensitivity of HSV-tk gene transfected HeLa cells to different concentrations of GCV application the MTT method.Results HeLa cells that transfected with the two kinds of plasmid,was treated with different concentrations of doxycycline,after 48 h,RT-PCR results show that HSV-tk gene expression increased gradually with the increase of concentration of doxycycline,the expression level reached the peak in the doxycycline for 4 μg/ml.when added by doxycycline,the cell sensitivity to GCV was increased.Conclusion HSV-tk gene expression and antitumor function was successfully regulated used tetracycline in HeLa cells then provides experimental and theoretical basis for further explore expression regulation of suicide gene during antitumor.