| Hepatomacellular carcimoma (HCC) is one of the most common malignanciesworldwide with an extremely poor prognosis. The major etiologic risk factors for HCCdevelopment include toxin, hepatitis B virus (HBV) and hepatitis C virus(HCV) infectionas well as various inherited metabolic disorders. Surgical treatment is the most importanttreatment of HCC. However, clinical observation have shown that tumor recurrence ratesare very high in patients with HCC who receive medical or surgical treatment. Thus newtreatment modalities must be pursued. With the expectation of increasing therapeuticefficacy, gene therapy has been introduced as a new direction in treatment for HCC. Genetherapy strategies against HCC include suppressor gene therapy, anti-sene gene therapy,immunogene therapy, suicide gene therapy and combinanted-gene therapy. However, theoverall results of the studies are still disappointing. How to search for tumor-specificagent of gene therapy? How to achieve appropriate spatial and temporal control of theexpression of the therapeutic gene? How to construct tumor tissue-targeted vector? Thesequestions are still critical problems in tumor gene therapy. Adenovirus is a medium-sized, icosahedral virus that contained a double-strandedlinear DNA genome. Adenovirus has many advantages include broad tissue tropism,stability, safety, high lever expression and replication-defective(E1A and E1B deletion) 5adenoviral vector can accommodate up to 7.5kb of foreign genes and can be amplified tohigh titers in 293 cells. HIV-1 vpr is a 96-a.a. 14kDa protein associated with the HIVvirus particle. HIV-1 vpr has been shown to affect tumor cells in ways similar to that ofp53 and other tumor suppressor gene. Several laboratories have reported that vpr inducesapoptosis following induction of G2/M cell cycle arrest in some tumor lines. Pang et al.reported that the mice of which the primary tumor were completely regressed by the vprwere additionally protected from a secondary challenge of tumor cells. These resultssuggest that the unique biological properties of vpr shown in some tumor lines suggestthat vpr may be a useful biological agent for anti-cancer therapy. Many cancers oftenreexpress fetal or embryonic genes, and AFP gene expression is reactivated in HCC cells.So we can use AFP promoter drive foreign gene expression to target AFP-producinghepatoma cells to achieve hepatoma-specific gene therapy. Targeting of tumor cells is crucial for gene therapy of malignant diseases. This canbe achieved by tumor-targeted gene transfer or tumor-specific gene expression. Base onabove consideration and theory, we use Ad5 pAdEasy system to generate recombinantadenovirus by exploiting E.coli BJ5183 homologous recombination machinery. Weconstructed a replication-defective adenoviral vector expressing vpr gene drived by AFPpromoter to achieve HCC-targeted gene therapy, named by rvAdAFP-vpr. We confirmedthat vpr gene and AFP promoter gene have been integrated into adenoviral genome byPCR and Southern Blot methods. In the present our study, non-AFP-producing hepatoma cell SMMC-7721 andAFP-producing hepatoma cell BEL-7402 infected with rvAdAFP-vpr and the expressionof vpr protein in the infected cells was examined at 96h post infection. By flowcytometer analysis, we found that vpr protein special expressed in the AFP-producinghepatoma cell BEL-7402 and arrested BEL-7402 cell cycle at G2 phase, and we didn't 6find that expression of vpr protein and G2 cell cycle arrest in non-AFP-producinghepatoma cell SMMC-7721. At the same time, we found that vpr protein could induceBEL-7402 cell apoptosis by confocal microscopy. We made use of flow cytometer toanalysis activation of apoptosis-associated protein Bax, Caspase 3, Caspase 9, Caspase 8and cytochrome c. Our results showed that high level Bax expression and activation ofCaspase 3, Caspase 9 and cytochro...
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