摘要
BACKGROUND:The incidence of hepatocellular carcinoma (HCC)in China is closely related to the population infected with hepatitis B virus(HBV).HCC cells with HBV secrete soluble HBsAg into blood but do not express it on the cell membrane This study aimed to construct and investigate a new glycosyl phosphatidylinositol(GPI)-anchored protein(GPC3+α+EGFP) as a DNA vaccine against HCC associated with HBV. METHODS:A recombinant plasmid(pcDNA3.1(+)/GPC3+ α+EGFP)was constructed and verified by restriction endo nuclease digestion and sequencing.pcDNA3.1(+)/GPC3+α+ EGFP was transfected into HepG2 cells(experimental group) using lipofectamine 2000.pEGFP-N1-transfected HepG2 cells were used as a negative control,and non-transfected HepG2 cells sreved as a blank control.HepG2 cells that steadily expressed the fusion protein GPC3+α+EGFP were screened by G418,propagated,and co-cultured with lymphocytes from healthy donors.Cell proliferation was measured by the classic sulforhodamine B assay.Apoptosis was assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL),and Fas gene transcription was determined by quantitative fluorescent PCR. RESULTS:The pcDNA3.1(+)/GPC3+α+EGFP plasmid was successfully constructed.In the experimental group,green fluorescence was observed at the cell periphery and in the cytoplasm,whereas in the negative control group,fluorescence was evenly distributed throughout the cell.Proliferation of the experimental group significantly decreased after 72 hours compared to the negative and blank control groups.Furthermore,the number of apoptotic cells was statistically different among the three groups as determined by a contingency table Chisquare test;the experimental group had the highest incidence of apoptosis.Fas gene transcription in the experimental group was higher than in the two control groups,and an increasing trend with time in the experimental group was observed. CONCLUSION:A chimeric,membrane-anchored protein, GPC3+α+EGFP,localized to the membrane of HepG2 cells and inhibited proliferation and accelerated apoptosis through a Fas-FasL pathway after co-cultivation with lymphocytes.
BACKGROUND:The incidence of hepatocellular carcinoma (HCC)in China is closely related to the population infected with hepatitis B virus(HBV).HCC cells with HBV secrete soluble HBsAg into blood but do not express it on the cell membrane This study aimed to construct and investigate a new glycosyl phosphatidylinositol(GPI)-anchored protein(GPC3+α+EGFP) as a DNA vaccine against HCC associated with HBV. METHODS:A recombinant plasmid(pcDNA3.1(+)/GPC3+ α+EGFP)was constructed and verified by restriction endo nuclease digestion and sequencing.pcDNA3.1(+)/GPC3+α+ EGFP was transfected into HepG2 cells(experimental group) using lipofectamine 2000.pEGFP-N1-transfected HepG2 cells were used as a negative control,and non-transfected HepG2 cells sreved as a blank control.HepG2 cells that steadily expressed the fusion protein GPC3+α+EGFP were screened by G418,propagated,and co-cultured with lymphocytes from healthy donors.Cell proliferation was measured by the classic sulforhodamine B assay.Apoptosis was assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL),and Fas gene transcription was determined by quantitative fluorescent PCR. RESULTS:The pcDNA3.1(+)/GPC3+α+EGFP plasmid was successfully constructed.In the experimental group,green fluorescence was observed at the cell periphery and in the cytoplasm,whereas in the negative control group,fluorescence was evenly distributed throughout the cell.Proliferation of the experimental group significantly decreased after 72 hours compared to the negative and blank control groups.Furthermore,the number of apoptotic cells was statistically different among the three groups as determined by a contingency table Chisquare test;the experimental group had the highest incidence of apoptosis.Fas gene transcription in the experimental group was higher than in the two control groups,and an increasing trend with time in the experimental group was observed. CONCLUSION:A chimeric,membrane-anchored protein, GPC3+α+EGFP,localized to the membrane of HepG2 cells and inhibited proliferation and accelerated apoptosis through a Fas-FasL pathway after co-cultivation with lymphocytes.
基金
supported by grants from the NaturalScience Foundation of China(30500239)
the China PostdoctoralScience Foundation(20060400227)
