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Objective: To construct a recombinant lentivirus RNA interference (RNAi) vector carrying hTERT gene, and to obtain the titer of the lentiviral stock for investigating the expression in the eukaryotic cells and the effect on the hTERT gene silencing in the eukaryotic cells., Methods: Two complimentary oligos of small interference RNA (siRNA) with hairpin structures targeting the hTERT gene and a negative control were synthesized, then ligated with pLVTHM vector and sequenced. The recombinant vectors were then transfected with viral packaging mix into T293 cells, viral supernatant was harvested to determine the titer. U87 cells infected by virus were harvested and the expression of hTERT, telomerase activity and apoptosis were detected by reverse transcription-PCR(RT-PCR), TRAP assay and flow cytometry separately., Results: Sequencing data showed that the constructed plasmids contained the correct sequences of hTERT siRNA transcript templates. A vector producing cell line T293 was established, and the titer for transfection was obtained. RT-PCR and TRAP flow cytometry analyses demonstrated that hTERT shRNA expression construct could suppress the expression of hTERT and telomerase activity and induce apoptosis., Conclusion: A lentivirus RNAi vector targeting hTERT gene was successfully constructed, which decreased the expression of hTERT and telomerase activity effectively and induced apoptosis. It has set up a research platform for the gene therapy of tumors which take hTERT as the target.

Objective: To study inhibitory efficacy of combined gene therapy for malignant gliomas transfected with antisense human telomerase reverse transcriptase (hTERT)/PTEN in vitro and in vivo., Methods: To construct two adenovirus recons which contained antisense hTERT and wild-type PTEN respectively with high performance homologous recombination system in bacteria. The two adenovirus recons were transfected into U251 human malignant glioma cells combinedly or respectively in vitro and in vivo. U251 cell proliferation in vitro was determined by MTT assay and flow cytometry, tumor growth in vivo was measured by the volume of glioma in nude mice. Telomerase activity was detected by telomeric repeat amplification protocol (TRAP) assay. Expression of hTERT and PTEN protein was detected by Western blotting methods., Results: After transfection in vitro, the growth of U251 cells was inhibited significantly. The inhibitory effect was time-dependent. The strongest inhibition was observed in combined transfection group, at the 6th day, the survival rate was 37.6%, telomerase activity (only 28.8TPG) was inhibited significantly, hTERT protein expression was inhibited significantly too, which was 0.2106, but PTEN protein expression was increased significantly, which was 0.9630. In vivo, the growth of tumors was also effectively inhibited., Conclusion: Growth of malignant glioma cells is effectively inhibited after transfection with combined antisense hTERT and PTEN in vitro and in vivo.