Construction Of Expression Vector Containing HSV-tk Gene And In Vitro Study On Killing Effects Of HSV-tk/GCV System On Human Hepatocellular Carcinoma Cell Line SMMC-7721

Objective: The aim of this study was to construct retroviral expression vector containing HSV-tk sucide gene. We evaluated the killing effects of HSV-tk/GCV on human hepatocellular carcinoma cell line SMMC-7721 in vitro. Methods: HSV-tk gene was subcloned into retroviral vector pLXSN by recombinant DNA technology. pL(tk)SN was packaged with PA317 and selected in G418 to obtain the positive clones, which was able to produce stable retrovirus. SMMC-7721 was infected by the recombinant retrovirus. The positive clones were obtained after G418 selection and were termed SMMC/tk . The integration and expression of tk gene in SMMC/tk cells were identified by PCR and RT-PCR.The GCV killing effects on gene-modified cells were assessed by observing growth state of SMMC/tk, MTT and FCM. Results: By sequencing , restriction digestion and PCR we confirmed that the sequence, length, position and orientation of inserted tk gene was correct. Then target vector pL(tk)SN was constructedsuccessfully. The retroviral vector pL(tk)SN was generated and stable titer producing cell C34 was established(1.8x106/ml). PCR and RT-PCR demonstrated that tk gene was integrated into the SMMC/tk genome and expressed at the mRNA level. There was no significant difference in cell proliferation among SMMC/tk, SMMC/0 and SMMC-7721 cells. SMMC/tk cells were highly sensitive to GCV especially in low concentration of GCV. Conclusions: We successfully constructed expression vector containing HSV-tk gene .In vitro study showed that hepatocellular carcinoma cell line SMMC-7721 was sensitive to HSV-tk/GCV system . These results suggest that HSV-tk/GCV system is an effective gene therapy and will lay some foundation for clinical treatment of hepatocellular carcinoma.