AIM: To determine the lethiferous effects of a recombinant vector carrying thymidine kinase (TK) suicide gene on 2.2.15cells and the possible self-modulating mechanism.METHODS: A self-modulated expressive plasmid pcDN...AIM: To determine the lethiferous effects of a recombinant vector carrying thymidine kinase (TK) suicide gene on 2.2.15cells and the possible self-modulating mechanism.METHODS: A self-modulated expressive plasmid pcDNA3-SCITK was constructed by inserting the fragments carrying hepatitis B virus antisense-S (HBV-anti-S) gene, hepatitis C virus core (HCV-C) gene, internal ribosome entry site (IRES) element of HCV and TK gene into the eukaryotic vector pcDNA3, in which the expression of TK suicide gene was controlled by the HBV S gene transcription. 2.2.15cells that carry the full HBV genome and stably express series of HBV antigen were transfected with pcDNA3-SCITK or vector pcDNA3-SCI which was used as the mock plasmid. The HepG2 cells transfected with pcDNA3-SCITK were functioned as the negative control. All the transfected cells were incubated in DMEM medium supplemented with 10 μg/ml. Of ganciclovir (GCV). The HBsAg levels in the supernatant of cell culture were detected by ELISA on the 1st, 3rd and 6th day post-transfection. Meanwhile, the morphology of tranfected cells was recorded by the photograph and the survival cell ratio was assessed by the trypan blue exclusion test on the 6th day posttransfection.RESULTS: The structural accuracy of pcDNA3-SCITK was confirmed by restriction endonuclease digestion, PCR with specific primers and DNA sequencing. The HBsAg levels in the supernatant of transfected 2.2.15 cell culture were significantly decreased on the 6th day post-transfection as compared with that of the mock control (P＜0.05). The lethiferous effect of pcDNA3-SCITK expression on 2.2.15cells was initially noted on the 3rd day after transfection and aggravated on the 6th day post transfection, in which the majority of transfected 2.2.15 cells were observed shrunken, round in shape and even dead. With assessment by the trypan blue exclusion test, the survival cell ratio on the 6th day post transfection was 95% in the negative control and only 11% in the experimental group.CONCLUSION: The results indicate that suicide gene expression of pcDNA3-SCITK can only respond to HBV-S gene transcription, which may be potentially useful in the treatment of HBV infection and its related liver malignancies.展开更多
基金the State Key Basic Research Program of China(973 Program,No.20014CB51008)National Key R & D Program of China for the 10~(th)Five-Year Plan Period(No.2001BA705B05)
文摘AIM: To determine the lethiferous effects of a recombinant vector carrying thymidine kinase (TK) suicide gene on 2.2.15cells and the possible self-modulating mechanism.METHODS: A self-modulated expressive plasmid pcDNA3-SCITK was constructed by inserting the fragments carrying hepatitis B virus antisense-S (HBV-anti-S) gene, hepatitis C virus core (HCV-C) gene, internal ribosome entry site (IRES) element of HCV and TK gene into the eukaryotic vector pcDNA3, in which the expression of TK suicide gene was controlled by the HBV S gene transcription. 2.2.15cells that carry the full HBV genome and stably express series of HBV antigen were transfected with pcDNA3-SCITK or vector pcDNA3-SCI which was used as the mock plasmid. The HepG2 cells transfected with pcDNA3-SCITK were functioned as the negative control. All the transfected cells were incubated in DMEM medium supplemented with 10 μg/ml. Of ganciclovir (GCV). The HBsAg levels in the supernatant of cell culture were detected by ELISA on the 1st, 3rd and 6th day post-transfection. Meanwhile, the morphology of tranfected cells was recorded by the photograph and the survival cell ratio was assessed by the trypan blue exclusion test on the 6th day posttransfection.RESULTS: The structural accuracy of pcDNA3-SCITK was confirmed by restriction endonuclease digestion, PCR with specific primers and DNA sequencing. The HBsAg levels in the supernatant of transfected 2.2.15 cell culture were significantly decreased on the 6th day post-transfection as compared with that of the mock control (P＜0.05). The lethiferous effect of pcDNA3-SCITK expression on 2.2.15cells was initially noted on the 3rd day after transfection and aggravated on the 6th day post transfection, in which the majority of transfected 2.2.15 cells were observed shrunken, round in shape and even dead. With assessment by the trypan blue exclusion test, the survival cell ratio on the 6th day post transfection was 95% in the negative control and only 11% in the experimental group.CONCLUSION: The results indicate that suicide gene expression of pcDNA3-SCITK can only respond to HBV-S gene transcription, which may be potentially useful in the treatment of HBV infection and its related liver malignancies.
