| Glioma is the most common malignant intracranial tumor with significant morbidity and mortality, accounting for40%-50%of primary brain tumor in adult. At present, the clinical therapeutic principles are mainly surgical resection of the lesion with postoperative radiotherapy and/or chemotherapy, which have made great progress, however could not substantially change the natural history of these lethal neoplasms. With the advancement in tumor molecular biological theory and technology, researches gradually develop about gene therapy of brain glioma. Although there are many methods and strategies of gene therapy, most of them are still in the experimental stage, and the outcomes of some clinical tests are not so satisfactory. Recently, researchers have perceived that brain glioma is a difficult controlling disease. The mechanism of its genesis is rather complex that involves many factors and steps. Thus, the monogene therapy has its drawbacks as well as advantages. So the research models from monogene to polygene, from one level to integrated levels, from the cells to the whole, have gradually become the main direction of tumor research in the future, and the combined or comprehensive tumor gene therapy turns to be the trend of research, too.Our group has been involving in the basic research of gene therapy of brain glioma for a long time. In previous study, we found that high level of basic fibroblast growth factor (bFGF) is associated with malignant degrade of glioma. bFGF abnormal highly expressed by neoplastic astrocytes stimulates the proliferation of astrocytoma cells and maybe inhibit glioma cell apoptosis. Meanwhile, we have studied the HIV-1viral protein R (VPR), and found that this protein could induce cell cycle G2arrest and apoptosis in glioma cells and inhibit the proliferation of glioma significantly in vitro. Based on the previous researches and following the design idea of combined gene therapy, in this study we select bFGF, which plays a key role in cell proliferation, as the target gene for RNA interference, and at the same time introduce exogenous gene HIV-1Vpr, which could induce cell apoptosis, into the glioma cells. This optimized therapeutic scheme consisting of RNA interference technology and exogenous gene transduction is thought to exert an optimal anti-tumor effect by regulating the growth and metabolism of tumor cells through two pathways: proliferation inhibition and apoptosis induction. And we also hope to summarize a new combined gene therapy strategy by this study.This study will be divided to3parts:In the first part, the recombinant adenoviruses of rAd5-bFGF-siRNA and rAd5-Vpr, which carry bFGF small interfering RNA and Vpr gene respectively, were constructed by the method of LR recombination. After PCR identification, they were amplified and the titers detected were about2.0X1010GTU/mL or5X108PFU/mL.The second part is an in vitro study. In this part we used the recombinant adenoviruses constructed in the first part and transfected U251, LN229and A172cell lines with them to study their roles in cell proliferation and apoptosis. First, we determined the transfection condition of adenovirus, i.e. at a multiplicity of infection (MOI) of50, then by MTT assay the proliferation activity of cells was detected, and the results revealed that both rAd5-bFGF-siRNA and rAd5-Vpr could inhibit the proliferation of glioma cells, and the combined treatment group was more efficient. Second, the apoptosis effect and the change of cell cycle were determined by Hoechst stain and flow cytometry (FCM) technology respectively. The results showed that rAd5-bFGF-siRNA had no effect on cell cycle while rAd5-Vpr could induce cell cycle G2arrest, and both could induce cell apoptosis, with the combined group the most efficient, and the rAd5-Vpr group more efficient than the rAd5-bFGF-siRNA group. At last, the methods of immunofluorescence, RT-PCR and Western blot were performed to detect the expression of target genes in different levels. Furthermore, we had preliminarily investigated the mechanisms of rAd5-bFGF-siRNA and rAd5-Vpr induced apoptosis in glioma cells by determination of the expression amounts of relevant apoptotic proteins, cell cycle proteins and signal pathway proteins. The results demonstrated that the Bcl-2, Bcl-xL and Cyclin D1proteins showed decreased expression while the Caspase-3and Fas-L proteins increased expression in either bFGF-siRNA group or Vpr group. The trends were more obvious in the combined treatment group which suggested that cell apoptosis could be induced by both bFGF gene silencing and Vpr gene transduction, and the combined group has an enhanced effect. In addition, the Aktl protein did not show any change in all groups which suggested that the rAd5-bFGF-siRNA and rAd5-Vpr induced cell apoptosis might not through the EGFR-PI3K-Akt/PKB pathway. And Stat3could be downregulated in the rAd5-bFGF-siRNA transfected groups, suggesting that Jak/STAT pathway might play a role in the bFGF depletion-triggered apoptosis. Moreover, Bax increased significantly in the bFGF-siRNA group while not the same in the combined treatment group, which might also suggest that the expression of Bax was impacted by both bFGF and Vpr gene, and they seemed not to have a synergistic effect. The detailed mechanisms need to be under further investigations.The third part is an in vivo experiment. First, we constructed the models of LN229glioma in nude mice subcuteously and divided them into normal control group, mock group, bFGF-siRNA group, Vpr group and combined treatment group. There were6mice in each group and they were either untreated or treated with PBS, rAd5-bFGF-siRNA, rAd5-Vpr and rAd5-bFGF-siRNA plus rAd5-Vpr, respectively. The tumor volumes were dynamically observed to draw a growth curve. HE, immunohistochemical and TUNEL staining were performed to view cell morphology, detect the changes of relevant target proteins and cell apoptosis respectively. Also the ultrastructural changes were observed by electron microscope. All of above methods used were to study the in vivo anti-tumor and apoptotic effects of the recombinant adenoviruses. The outcomes showed that both rAd5-bFGF-siRNA and rAd5-Vpr could exert an inhibition effect on tumor, which was similar to that of in vitro experiments.In a word, from this study we can conclude:Both bFGF gene silencing and Vpr gene transduction could inhibit proliferation and induce apoptosis in glioma cells in vitro and in vivo, and the combined treatment group showed a reinforcing effect. |
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