| Lung cancer is a common malignant tumor of respiratory system, and its incidence and mortality are increasing rapidly year after year. Although active surgical treatment and radio-chemotherapy have been conducted in clinic, the curative effect and prognosis of the disease are still disappointed, up to now, partially due to the difficult early diagnosis. Investigation of pathogenesis and new treatment of lung cancer has been carried out for decades. The result showed that as most solid rumors, growth and metastasis of lung cancer critically related to the angiogenesis. Among all of the known factors of tumor angiogenesis, vascular endothelial growth factor (VEGF) is the most important and direct one that can stimulate tumor angiogenesis. Conversely, inhibition of tumor angiogenesis can inhibit tumor growth and metastasis. Being sequence-specific binding and cleaving specific RNA of ribozyme, the target gene expression can be destructed by an artificially designed and synthesized ribozyme. Therefore, a great attention has been attracted into the filed of gene therapy for cancers with the ribozyme. Attempt of anti-tumor angiogenesis and high specific inhibition adverse gene by ribozyme is dramatically rising recent years, however, treatment with ribozyme to inhibit VEGF expression can not avoid inhibition of normal expression of VEGF in other5tissues and organs due to unable to deliver ribozyme gene site-specifically into the target organ. Adenovirus has been most widely used as a viral vector in gene therapy for being able to infect both dividing and non-dividing cells, high efficacy of gene transfer in many type of human cancers, easy to obtain in high liter and its natural tropism to respiratory epithelial tissue. So adenoviral vector may be particularly appropriate for the gene therapy of lung cancer.To investigate the influence of the VEGF165 expression and the biological characters of A549 cell as well as the inhibition of tumor growth by ribozyme, the ribozyme target VEGF165 mRNA site 212 and mutant ribozymes were designed and synthesized, and the adenovirial vector constructed. Then human lung cancer adenocarcinoma cell line A549 were infected with recombinant adenovirus containing ribozyme and mutant ribozyme to evaluate the effects of VEGF ribozyme on the angiogenesis of lung cancer by observation of tumor formation, tumor growth speed and tumor end volume.The main technic and results in this study consisted of:1. The hammerhead ribozyme target the messenger RNA site 212 of VEGF 165 (Rz212) and its corresponding mutant counterpart (mRz212) were designed by computer and synthesized based on VEGF 165 cDNA. After gene recombination, prokaryotic vectors containing Rz212 and mRz212 with 5', 3'c/s-ribozymes were constructed. The sequences are confirmed by restriction enzyme and sequencing analysis.2. Ribozyme, mutant ribozyme and target gene were transcripted by T7 RNA polymerase and tested for cleavage activity in vitro. After 6% denatural PAGE and autoradiography, the results were analyzed. Cleavage efficiency of ribozyme reached 90.7%, otherwise the mutant ribozyme was unable to cleave VEGF165 mRNA.3. Rz212 and mRz212 with 5', 3' c/s-ribozymes were subcloned into adenoviral shuttle plasmid pAdCMV, and formed transfer plasmids of pAdCMVRz212 and pAdCMVmRz212. Transfer plasmids constructed correctly confirmed by restriction enzyme. The transfer plasmid and adenoviral genome plasmid pJM17 cotransfected to 293 cells to package recombinant adeno virusparticles. RNA dot hybridization and PCR test indicated that recombinant adenoviruses containing Rz212 and mRz212 were produced. The liter of purified adenovirus was 8.2* 101-2 particles/ml.4. Human lung adenocarcinoma cell line A549 cells were infected with recombinant adenovirus. KNA dot hybridization proved that ribozyme and mutant ribozyme had been successfully transfected into A549 cells. Compared with control, empty vector and mutant ribozyme infected cells group, expression of VEGF165 in the ribozyme...
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