Empirical study of hepatocellular carcinoma's gene therapy using hTERTp-TK/GCV in HepG2 which was mediated by chitosan nanoparticles vehicles / 肿瘤研究与临床

ABSTRACT

Objective To compare the transfection efficiency of galactosylated chitosan nanoparticle vehicle with chitosan nanoparticles vehicle,and observe the therapeutic effect of pGL3-hTERTp-TK on HCC cell line HepG2. Methods Preparing the chitosan and galaetosylated chitosan. Constructing the pGL3-hTERTp-TK plasmid and the Ch/DNA and GC/DNA complexes.Transfecting the HepG2 and the normal hepatic cell L-02 with chitosan/DNA and galactosylated chitosan/DNA complexs.Detecting the fluorescence and the expression of luciferase gene using the fluorescent microscope and the scintillation counter.Detecting the cell growth and apoptosis through the Caspase-3 and the flow cytometry. Results Two clear straps appeared in the agarose gel.The locations were 300 bp and 1100 bp.The relative lueiferase activity of pGL3-hTERTp-Luc+mediated by galactosylated chitosan was powerful than which of pGL3-hTERTp-Luc+mediated by chitosan in HepG2 by the scintillation counter.However,the relative luciferase activity was very weak in L-02.The same results were observed by fluorescent microscope.When the concertration of the GCV was 10 μg/ml(t=51.40,P=0.000),the HepG2 cell inhibition which was transfected by GC-pGL3-hTERTp-TK was obviously different from the L-02 cell inhibition which was transfected by GC-pGL3-hTERTp-TK in the statistics.The significant apoptotic rate was 65.28%in HepG2 which was transfected with GC/DNA,whereas it was only 10.80% in L-02. The significant apoptotic rate in HepG2 which was transfected with GC/DNA was very higher than the other groups (LSD, P ＜0.05). The significant apoptotic rate (10.80%) in L-02 which was transfected with GC/DNA was very higher than the group which was Ch-pGL3-control (LSD, P =0.000). The average fluorescence intensity of the HepG2 which is transfected by the Ch-pGL3-hTERTp-TK was 168.02± 3.68. The average fluorescence intensity of the HepG2 which is transfected by the GC-pGL3-hTERTp-TK was 204.45 ±3.45. The two groups had a significant difference in the statistics (t=-12.504, P＜0.05). Conclusion Galactosylated chitosan has higher transfection efficiency than chitosan. GC-pGL3-hTERTp-TK could specially attack HCC cell line and almost has no influence on normal hepatic cells.