ABSTRACT
Objective: To construct a lentiviral vector (LV) of RNA interference (RNAi) targeting HBs gene and to observe its effect on the replication of HBV and expression of antigens. Methods: The effective sequence of siRNA targeting HBs gene was confirmed in our previous study. The complementary DNA containing both sense and antisense Oligo DNA of the targeting sequence was designed, synthesized and cloned into the lentivirus vector (pGCLM-GFP). The resulting lentivirus vector containing HBs shRNA was named LVshHBs, and it was confirmed by PCR and sequencing. 293T cells were cotransfected with lentivirus vector pGCLM-GFP, pHelper 1.0 and pHelper 2.0. All virus stocks were produced by calcium phosphate-mediated transfection. The titer of virus was tested according to the expression level of GFP. HepG2. 2.15 cells were infected with LVshHBs and the supernatant of the cells was subjected to ELISA, Western blotting analysis and HBV DNA quantitative analysis. Results: PCR and DNA sequencing demonstrated that the LVshHBs-producing HBs shRNA was successfully constructed. The titer of concentrated virus was 5 × 108-2 × 109 TU/ml. The inhibitory effect was efficient and the corresponding viral transcript and expression of antigens were decreased after infection. The inhibitory effect was observed 4 days after infection and peaked 9 days after the initial treatment with RNAi. Secreted HBsAg was reduced by > 70% in cell culture compared with the negative control, which is also confirmed by Western blotting and real time PCR. After quantification of HBV DNA, the level of DNA relative to the controls was also significantly reduced after RNAi treatment(P<0.05). Conclusion: The lentivirus RNAi vector of HBs has been successfully constructed. The lentiviral microRNA-based RNAi targeting HBs can specifically mediate the down regulation of HBs expression, inhibiting HBV replication and antigen expression.
ABSTRACT
<p><b>BACKGROUND</b>The Pringle maneuver, which has been the standard for hepatic resection surgery for a long time, has the major flaw of ischemic damage in the liver. The aim of this research was to evaluate hepatic blood inflow occlusion with/without hemihepatic artery control vs. the Pringle maneuver in hepatocellular carcinoma (HCC) resection.</p><p><b>METHODS</b>Two hundred and eighty-one cases of resection of HCC with hepatic blood inflow occlusion (with/without hemihepatic artery control) and the Pringle maneuver from January 2006 to December 2008 in our hospital were analyzed and compared retrospectively; among them 107 were in group I (Pringle maneuver), 98 in group II (hepatic blood inflow occlusion), and 76 in group III (hepatic blood inflow occlusion without hemihepatic artery control). The operation time, intraoperative blood loss, postoperative liver function and complications were used as the endpoints for evaluation.</p><p><b>RESULTS</b>The operative duration and intraoperative blood loss of three groups showed no significant difference; alanine aminotransferase, total bilirubin and incidence of postoperative complications were significantly lower in groups II and III postoperation than those in group I.</p><p><b>CONCLUSION</b>Hepatic blood inflow occlusion without hemihepatic artery control is safe, convenient and feasible for resection of HCC, especially for cases involving underlying diseases such as cirrhosis.</p>