title: [Inhibitory effect of lentivirus-mediated hTERTp-TK combined with hTERTp-tumstatin on human hepatocarcinoma HepG2 cells].
abstract: OBJECTIVE
  To observe targeted expression of recombinant lentivirus-mediated (Lv)-hTERTp-TK and Lv-hTERTp-tumstatin in HepG2 cells, and explore the inhibitory effect of their combination on HepG2 cells both in vitro and in vivo.
  
  
  METHODS
  Lv-hTERTp-TK and Lv-hTERTptumstatin were used to infect HepG2 and L02 cells at different MOIs. Transfection efficiency was observed by fluorescence microscopy. Expression of TK and tumstatin mRNA was detected by reverse-transcriptase PCR. Proliferation and apoptosis were detected by MTT and flow cytometry, respectively. The HepG2 cells were examined by real time-PCR and western blotting to determine expression level of bcl-2 and VEGF mRNA and protein.A murine hepatocellular carcinoma model was established by injecting 1 * 10(7) HepG2 cells into 30 BALB/c nude mice. The modeled mice were randomly divided into a control group, mock group, Lv-hTERTp-tumstatin group, Lv-hTERTp-TK group, and combination group for four weeks of injections at regular intervals of PBS, Lv-hTERTp-null, Lv-hTERTp-tumstatin, Lv-hTERTp-TK, and Lv-hTERTp-tumstatin plus Lv-hTERTp-TK, respectively.Changes in tumor volume and weight, and cell morphology of tumor and major organs, were assessed by hematoxylin-eosin staining.Microvascular density of tumor tissue and cell apoptosis were assessed by immunohistochemical and TUNEL staining, respectively.
  
  
  RESULTS
  The Lv-infected HepG2 cells, and not the Lv-infected L02 cells, expressed TK and tumstatin. Lv-hTERTp-TK and Lv-hTERTp-tumstatin, alone or in combination, inhibited proliferation and increased apoptosis of the HepG2 cells, but the combination was more effective than either alone (P less than 0.05). None of the treatments affected proliferation or apoptosis of the L02 cells (P more than 0.05). The combination also led to a greater reduction of bcl-2 and VEGF than either alone (all, P less than 0.05). Tumor growth was significantly inhibited by the combination (P less than 0.05). In vivo, the combination treatment induced the greatest amount of apoptosis of the HepG2 cells. Cell morphology of major organs such as liver, spleen and kidney were similar to the control group. The combination also produced the most significant effect on tumor microvascular density (P less than 0.05) and the highest apoptosis index (P less than 0.05).
  
  
  CONCLUSIONS
  The HTERT promoter can induce targeted expression of TK and tumstatin in HepG2 cells. Lv-hTERTp-TK combined with Lv-hTERTp-tumstatin can significantly inhibit proliferation and induce apoptosis of human HepG2 cells in vitro and in vivo, which may be related to down-regulation ofbcl-2 and VEGF.
authors.affiliation: Department of Gastroenterology, the Second Affiliated Hospital, Shanxi Medical University, Taiyuan 030001, China.
authors.forename: Yu-Xi
authors.initials: YX
authors.lastname: Meng
authors.forename: Xin
authors.initials: X
authors.lastname: Niu
authors.forename: Zhi-Hua
authors.initials: ZH
authors.lastname: Deng
mesh_terms: D000818:Animals; D017209:Apoptosis; D001324:Autoantigens / Q000235:genetics*; D006528:Carcinoma, Hepatocellular / Q000628:therapy*; D045744:Cell Line, Tumor; D024141:Collagen Type IV / Q000235:genetics*; D015536:Down-Regulation; D005434:Flow Cytometry; D018014:Gene Transfer Techniques; D015316:Genetic Therapy; D005822:Genetic Vectors; D056945:Hep G2 Cells; D006801:Humans; D016086:Lentivirus; D008113:Liver Neoplasms / Q000628:therapy*; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D009368:Neoplasm Transplantation; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D019098:Telomerase / Q000235:genetics*; D014162:Transfection; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism
chemical_list: D001324:Autoantigens; D024141:Collagen Type IV; D019253:Proto-Oncogene Proteins c-bcl-2; D042461:Vascular Endothelial Growth Factor A; C057112:type IV collagen alpha3 chain; D019098:Telomerase