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AIM:To determine the lethiferous effects of a recombinantvector 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 carryinghepatitis B virus antisense-S(HBV-anti-S)gene,hepatitisC virus core(HCV-C)gene,internal ribosome entry site(IRES)element of HCV and TK gene into the eukaryoticvector pcDNA3,in which the expression of TK suicide genewas controlled by the HBV S gene transcription.2.2.15cells that carry the full HBV genome and stably expressseries of HBV antigen were transfected with pcDNA_3-SCITKor vector pcDNA_3-SCI which was used as the mockplasmid.The HepG2 cells transfected with pcDNA_3-SCITKwere functioned as the negative control.All the transfectedcells were incubated in DMEM medium supplemented with10 μg/ml,of ganciclovir(GCV).The HBsAg levels in thesupernatant of cell culture were detected by ELISA onthe 1~(st),3~(rd)and 6~(th)day post-transfection.Meanwhile,themorphology of tranfected cells was recorded by thephotograph and the survival cell ratio was assessed bythe trypan blue exclusion test on the 6~(th)day post-transfection.RESULTS:The structural accuracy of pcDNA_3-SCITK wasconfirmed by restriction endonuclease digestion,PCR withspecific primers and DNA sequencing.The HBsAg levels inthe supernatant of transfected 2.2.15 cell culture weresignificantly decreased on the 6~(th)day post-transfection ascompared with that of the mock control(P<0.05).Thelethiferous effect of pcDNA_3-SCITK expression on 2.2.15cells was initially noted on the 3~(rd)day after transfectionand aggravated on the 6~(th)day post transfection,in whichthe majority of transfected 2.2.15 cells were observedshrunken,round in shape and even dead.With assessmentby the trypan blue exclusion test,the survival cell ratio onthe 6~(th)day post transfection was 95 % in the negativecontrol and only 11% in the experimental group. CONCLUSION:The results indicate that suicide geneexpression of pcDNA_3-SCITK can only respond to HBV-Sgene transcription,which may be potentially useful in thetreatment of HBV infection and its related livermalignancies.
AIM: To determine the lethiferous effects of a recombinant vector carrying thymidine kinase (TK) suicide gene on 2.2.15 cells and the possible self-modulating mechanism. METHODS: A self-modulated expressive plasmid pcDNA3-SCITK was constructed by inserting the fragments carrying hepatitis B virus (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.2.2.15 cells that carry the full HBV genome and stably expressseries of HBV antigen were transfected with pcDNA_3-SCITK vector vector pcDNA_3-SCI which was used as the mockplasmid. The HepG2 cells transfected with pcDNA_3-SCITKwere functioned as the negative 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 1 st, 3 ~ (rd) and 6 ~ ( th) d ay post-transfection.Meanwhile, theyorphology of tranfected cells was recorded by the photograph and the survival cell ratio was assessed by the trypan blue exclusion test on the 6th (th) day post-transfection. RESULTS: The structural accuracy of pcDNA_3-SCITK wasconfirmed by restriction endonuclease digestion, PCR with specific primers and DNA sequencing. HBsAg levels inthe supernatant of transfected 2.2.15 cell culture weresignificantly decreased on the 6 ~ (th) day post-transfection ascompared with that of the mock control (P <0.05). Thelethiferous effect of pcDNA_3-SCITK expression on 2.2.15 cells was initial noted on the 3 ~ (rd) day after transfection and aggravated on the 6 ~ (th) day post transfection, in which the majority of transfected 2.2.15 cells were observedshrunken, round in shape and even dead. Worth assessmentby the trypan blue exclusion test, the survival cell ratio onthe 6 th (th) day post transfection was 95% in the negative control and only 11% in the experimental group. CONCLUSION: The results indicate that suicide geneexpression of pcDNA_3-SCITK can only respond to HBV-Sgene transcription, which may be potentially useful in the treatment of HBV infection and its related liver malignancies.