| Ribozymes are unique RNA molecules with enzymatic activity which is able to cleave targeted RNA substrates site-specifically. It has been identified that ribozymes are effective modulators of gene expression,such as activated oncogene or foreign virus gene. In comparison with antisense RNA,ribozymes can not only block but also cleave target RNAs. Moreover,they cleave the RNAs in a recycling manner,which means the ribozyme can dissociate from the ribo-RNA complex and bind to a new RNA molecule after cleaving one target molecule. It has been proved that the secondary structure of ribozymes is more stable and simpler than single-strand antisense RNA,which makes them more insensitive to the nuclease. In addition,ribozymes can also be artificially designed. For all these reasons,the discovery of the ribozymes provide more specific methods of gene therapy on the human cancer.At present ribozymes commonly used in gene therapy are hammerheadand hairpin types. Within them,the working conditions of the hairpin ribozymes are similar to normal human physical environment,whose cleavage activity is higher than hammerhead ones. All these make the hairpin ribozyme a more promising molecule for blocking the gene expression in vivo.The growth,development and migration of the cancer cells require vascular formation. Vascular endothelial growth factor(VEGF) is one of the most important factors acting upon vascular formation. It can effectively and specifically stimulate the vascular endothelial cells to enhance the formation and growth of the blood vessel,giving off intravascular substances,providing substrate for the migration of vascular endothelial cells and tumor cells. Ovarian cancer is one of the commonest gynecological malignancy with the highest death ratio which is difficulty to be diagnosed in early stages. Recent studies revealed that the VEGF and VEGF-receptor were overexpressed in ovarian cancer,and their expression levels were closely related to the prognosis of the patients. So it may be necessary to block the overexpression of the VEGF in tumor cells with ribozyme technique,thus to prevent the cells from over proliferation,and search for a new treatment of the ovarian cancer. In this study,we performed the following experiments:1. A 576bp VEGF gene was cloned with RT-PCR,construct pUC19-VEGF(+) and pcDNA3-VEGF(-) vectors. Enzyme digestion suggested the pUC19-VEGF(+) and pcDNAS-VEGF(-) were correct. DNA sequencing showed the cloned VEGF gene was as same as the VEGF sequence in human gene bank.2. Immunohistochemisty showed that the expressions of the VEGF and KDR in malignant and borderline tumor were significantly higher than those of benign tumor (P<0.05). KDR expressed not only in the tumor vascular epithelial cells but also in tumor cells. Expression of VEGF in ovarian cancers with lymphcyte migration showed significant differences in comparison with those without lymphcyte migration (P<0.05). These resultsindicated that VEGF and its receptor may play important roles in thedevelopment of the ovarian cancer.3. The anti-VEGF mRNA hairpin ribozyme was designed with the aid of a computer software. The secondary structure analysis showed this ribozyme possessed ideal cleavage site,and the homology analysis with other molecules in gene bank showed there were no other mRNA contained the complementary sequence to the ribozymes.4. The ribozyme gene was successfully inserted into the eukaryotic expression vector pcDNA at the EcoRI and BamHl sites. Enzyme digestion showed a 56bp ribozyme fragment,and the DNA sequencing indicated the synthesized and cloned ribozyme gene was correct.5. The VEGF mRNA and RZ were transcribed from the linealized DNA templates. Cleavage reaction showed specific cleaving activity of the ribozyme,which made the in vivo study of the ribozyme possible.6. The pcDNA3-RZ was transfected into the human ovarian cancer cells with the lipofectamine,and the RNA dot hybridization conformed the expression of ribozyme gene. The level of VEGF mRNA decreased dramatica...
|
PDF Full Text Request