| Renal cell carcinoma (RCC)is one of the most common cancer, RCC are solid tumours that arise from the proximal convoluted tubules of the kidney and are characerized by abundant neovascularization and arterio-venous fistula formation. Tumour vascularity is related to clinical outcome because metastases are more likely in patient with highly vascularized RCC. Widespread hematogenous dissemination occurs with metastatic disease, and secondary deposits exhibit similar hypervascularity to the primary tumour.Many observations show that angiogenesis plays an important role in the growth, progression, and metastasis of solid tumour, several angiogenic factors have been identified including vascular endothelial growth factor (VEGF) basic fibroblast growth factor (bFGF), transforming growth factor β KTGFβ 1), and so on. One important factor is vascular endothelial growth factor(VEGF), which is a specific mitogen for endothelial cells. VEGF promotes angiogenesis by stimulating capillary proliferation, migration, and permeability , and plays an important role in neovascularization of various neoplasms. The overexpression of VEGF has been demonstrated in many types of cancers, including colon cancer, breast cancer, lung cancer, ovarian cancer, and bladder cancer. As solid tumour with abundantneovascularization, the overexpression of VEGF has also been demonstrated in the RCC. Many studies have clearly demonstrated that the overexpression of VEGF is associated with the histologic grade and stage of the RCC.So the therapeutic approaches aimed at inhibiting the expression of VEGF can inhibit the growth of RCC. We construct mammalian expression vector carrying human VEGF antisense cDNA and to study the affection on VEGF expression and growth of renal cell carcinoma. Our study can provide some experimental bases for the gene therapy of RCC. Method:1. RT-PCR for VEGF, VEGF antisense cDNA was inserted into mammalian expression vector pcDNA3. 1, This vector contains a constitutive cytomegalovirus enhancer-promoter and a neomycin gene for selection of geneticin resistant colonies. Next using restrict enzyme to confirm the result by sequencing.2. The 786-0 human renal cell cancer cell-line was maintained in RMPI1640 medium supplemented with 10% fetal bovine serum. Colonies expressing pcDNA3. 1- (antisense) VEGF and pcDNA3. 1 were produced after transfection of pcDNA3. 1- (antisense) VEGF or pcDNA3. 1 without insert (empty vector) using Lipofect AMINE .Transfected colonies were selected in the presence of 700μg/ml G418. After selection, the two resistant colony to G418 named 786-0- (antisense) VEGF and 786-0-PC were chose.3. Using RT-PCR method to detect the expression of VEGFmRNA in the 786-0- (antisense) VEGF cell and 786-0-PC cell and 786-0 cell.4. Using immunohistochemical method to detect the protein expression of VEGF in the 786-0- (antisense) VEGF cell and 786-0-PC cell and 786-0 cell.5. Using MTT method to study growth of the 786-0- (antisense) VEGF cell and 786-0-PC cell and 786-0 cell.Result: Antisense VEGFcDNA was gained by RT-PCR, recombiant antisense VEGFmammalian expression vector pcDNAS. 1 was constructed . Compared with 786-0-PC cell groups and 786-0 cell groups, the amount of VEGFmRNA expression are decreased in the 786-0- (antisense) VEGF cell. Their difference is significant ( P< 0.01 ) .There is no significant between 786-0-PC cell groups and 786-0 cell groups in the the amount of VEGFmRNA expression (P > 0. 05). Compared with 786-0-PC cell groups and 786-0 cell groups, the amount of VEGF protein expression are lower in the 786-0-(antisense) VEGF cell groups. Their difference is significant ( P< 0. 01 ). There is no significant between 786-0-PC cell groups and 786-0 cell groups in the the amount of VEGF protein expression (P > 0. 05) . The growth of 786-0- (antisense) VEGF cells are slower than that of 786-0-PC cells and 786-0 cells.Conclusion : Antisense VEGF mammalian expression vector pcDNA3. 1 was constructed . The antisense VEGF gene can greatly decrease the VEGF...
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