Inhibitory Effect Of Antisense Human Telomerase RNA(hTR) Gene Mediated By Retroviral Vector On The Hepatocellular Carcinoma Cells.

Objective: To study the effects of retrovirus-mediated antisense hTR gene therapy on hepatocellular carcinoma cells and to investigate the significance of suppressing telomerase activity in the treatment of hepatocellular carcinoma.Methods: The procedure of plasmid extraction were routinely carried out, and the concentration and purity of the extracted plasmids were measured by UV spectrophotometer. Sense and antisense hTR gene were transfected into the packaging cell line PT67 by electroporation, and selected with G418 to get stable transfected cell lines. The recombinant retroviral supernantants were collected, filtered through 0.45μm neddles filters, and concentrated 100-fold by centrifugation. The virus in the supemants were titrated by transfecting NIH3T3 cell line. Then they were transfected into HepG2 cells. After G418 selection, PCR was resorted to demonstrate the successful integration of the hTR gene. Cell growth curves were drawn using MTT assay,meanwhile, the synergetic inhibitory effect of antisense hTR gene therapy and chemotherapeutic drugs(CDDP and 5-FU) to hepatocellular carcinoma cells were analyzed by MTT assay; and the expression of PCNA was determined by immunofluorescence; TRAP-PCR -ELISA was adopted to detect the telomerase activity; Cell cycle and cell apoptosis rate were evaluated by flow cytometry (FCM).Results: The expression of hTR gene could be amplified in HepG2-hTR-ï¿¡coRI and HepG2-hTR-5awHI, but not in untransfected HepG2 cells. It suggested that the sense and antisense hTR gene had been successMly intergrated into the genomic DNA of infected HepG2 cells. Data indicated that the antisense hTR complementary to the template region of telomerase was sufficient to inhibit cell growth and proliferation activity of HepG2 cells, the cell growth curve of HepG2-hTR-BamHI cell line was relatively mild compared with the other two control groups, the joint application of antisense hTR gene and chemotherapeutic drugs can inhibit the proliferation activity of HepG2 cells synergeticly; Compared with hTR sense clones and parental HepG2 cells, the fluorescence intensity of hTR antisense clones was weaker, and the positive rate was lower; Telomerase activity in the antisense hTR gene-treated group decreased significantly; FCM showed that the apoptosis rate of the experimental group increased, the proportion of HepG2 cells in G2/M phase was higher and the proportion of cells in S phase was lower compared with the control groups, but no obvious difference of G₀/G₁ phase distribution was found.Conclusion: Telomerase RNA is an important component of telomerase,down-regulation of its expression through anti-technology can lead to the growth inhibition and apoptosis induction of hepatocellular carcinoma cells. It can also improve the chemosensitivity of hepatocellular carcinoma cells, accordingly reducing drug consumption and drug side-effects, and is thought to be an important target of anti-tumour therapy.