双效逆转录载体的构建和受控自杀基因在乳腺癌细胞中表达的研究

目的：构建含有Tet-On基因调节系统及自杀基因HSVtk的重组逆病毒载体，研究其在乳腺癌细胞中HSVtk基因的受控表达。方法：用PCR方法扩增Tet-On基因，将其插入pRevTRE／HSVtk，形成重组载体pRevTRE／HSVtk／Tet-On。用脂质体介导法将pRevTRE／HSVtk／Tet-On导入乳腺癌细胞株(MCF-7)。经过HygromycinB筛选建立了一株稳定的受四环素衍生物强力霉素(Doxcycline，Dox)调控表达HSVtk基因的乳腺癌细胞株MCF／TRE／HSVtk／Tet-On。用RT-PCR的方法检测不同Dox浓度下HSVtk基因的表达，以MTT法检测不同Dox浓度下Ganciclovir(GCV)对乳腺癌细胞的杀伤作用。结果：成功构建了pRevTRE／HSVtk／Tet-On逆病毒载体。筛选出MCF／TRE／HSVtk／Tet-On细胞株，该细胞能在Dox的诱导下表达HSVtk基因并被GCV杀伤。结论：HSVtk基因产物可以将无毒性的Ganciclovir(GCV)转变成一种有毒的代谢产物，杀死乳腺癌细胞。而该基因的表达受到Tet-On调控，由强力霉素调节HSVtk基因表达。

The Construction of double-effective retroviral vector and the expression of HSVtk regulated by Tet-On in human breast cancer cells

Objective To construct retrovirus vector containing Tet-On gene regulation system and HSVtk gene and to detect the expression of HSVtk under the control of Tet-On regulation system in MCF-7 cells. Methods HSVtk gene was inserted into the plasmid pRevTRE/HSVtk and the recombinant retrovirus vector pRevTRE/HSVtk/Tet-On was constructed. Transfection of pRevTRE/HSVtk/Tet-On into MCF-7 cells was performed by using lipofectin. After selected by Hygromycin B, MCF-7 cell line stably expressing Tet-regulated HSVtk gene was established. mRNA level of pRevTRE/HSVtk/Tet-On at different Doxcycline (Dox) concentration was detected by RT-PCR, and MTT method was performed to detect the cell survival rate of pRevTRE/HSVtk/Tet-On at different Dox concentration when GCV was presented. Results Retrovirus vector, which contained Tet-On gene regulation system and HSVtk gene, was constructed successfully. MCF-7 cell line stably expressed tet-regulated HSVtk gene was established. The MCF-7 cells could be killed by GCV when the HSVtk gene was expressed under the regulation of Dox. Conclusion the product of HSVtk gene can convert the nontoxic GCV into toxic product and kill the breast cancer cells. The expression of HSVtk gene was controlled by Tet-On regulation system under the inducement of Dox.