ABSTRACT
Aim To construct a lentiviral vector for stable delivery of the connexin32 (Cx32) gene in human hepatocellular carcinoma cell line Huh7,and also to detect its effect on cell proliferation.Methods Human Cx32 gene sequence was obtained by whole gene synthesis and amplified by PCR,and was then inserted into LV5-GFP lentiviral vector to construct the recombinant plasmid LV5-GFP-hCx32.Following identified by restriction endonuclease digestion and DNA sequencing,this plasmid together with lentiviral package plasmid was transfected into 293T cells to produce the lentiviral particles,and the viral titer was assessed under fluorescent microscope.Targeted Huh7 cells were infected with the lentivirus (LV5-hCx32 and LV5-NC),and the infected cells after selection with puromycin were amplified and cultured.The expression and localization of Cx32 were detected by real-time RT-PCR,Western blot and immunofluorescence assay,respectively.The gap junction (GJ) function between adjacent cells was measured by dye transfer assay.The Huh7 cell proliferation capacity was determined by MTT and colony formation assays.Results The results of double enzyme digestion and DNA sequencing proved that the recombinant lentiviral vector LV5-GFP-hCx32 was successfully constructed.After packing in 293T cells,the recombinant lentivirus LV5-GFP-hCx32 with virus drops to 3 × 1011 TU · L-1 was obtained.Huh7 cells transiently infected with the lentivirus LV5-GFP-hCx32 remarkably over-expressed Cx32 at both mRNA and protein levels.Moreover,Cx32 expression was also significantly up-regulated in stably transfected Huh7 cells,and the presence of enhanced functional GJ in intact cells could be detected due to an increased amount of Cx32 protein along the plasma membrane at cell-cell contacts.Compared to LV5-NC group,the proliferation ability in Huh7 cells with recombinant Cx32 declined (P ＜ 0.05).Conclusions The lentiviral vector over-expressing Cx32 gene is successfully constructed,and can stably transfect Huh7 cells to yield sustained over-expression of exogenous Cx32 gene,thus eventually inhibits cell proliferation.
ABSTRACT
<p><b>OBJECTIVE</b>To evaluate the efficacy and safety of weekly paclitaxel combined with S-1 or fluorouracil in the first line treatment of advanced gastric carcinoma.</p><p><b>METHODS</b>Two hundred and forty patients with untreated advanced gastric carcinoma were randomized into two arms, patients in the experimental arm were given paclitaxel and S-1, while those in the control arm received paclitaxel and fluorouracil. The regimen of experimental arm was paclitaxel 60 mg/m(2) by intravenous infusion, day 1, 8, 15; S-1 80 - 120 mg/day given by oral administration, day 1 - 14. The regimen of control arm was fluorouracil 500 mg/m(2) by intravenous infusion continuously, day 1 - 5; CF 20 mg/m(2) by intravenous infusion, day 1 - 5. The regimens in both arms were repeated every 28 days. The efficacy and safety of both arms were assessed.</p><p><b>RESULTS</b>Two hundred and twenty-eight patients were analyzed in the full analysis set, and 192 patients were analyzed in per-protocol set (experimental arm 100 patients, control arm 92 patients). The overall response rates of experimental and control arms were 50.0% and 28.3% (P = 0.002), and the disease control rates were 82.0% and 70.7% (P = 0.064), respectively. The primary endpoints of experimental arm were non-inferior to that of the control arm. The secondary endpoint of experimental arm in terms of median progression free survival was significantly better than that of control arm (5 months versus 4 months, P = 0.006). The experimental arm had a higher incidence of grade III-IV bone marrow suppression than the control arm, but the incidence of fever in both arms was not significantly different.</p><p><b>CONCLUSIONS</b>Oral administration of S-1 is an alternative option of venous infusional fluorouracil. Weekly paclitaxel combined with S-1 is a safe regimen and has a promising efficacy.</p>