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Apoptotic protein inhibitor family is an important class of anti-apoptotic factor, the common characteristics of the structure of the family is the N-terminal containing one or more series baculovirus IAP repeat sequences end C contains or does not contain a RING finger . Survivin is an important member of the IAP family, has a strong anti-apoptotic role, and in the maintenance of cell mitosis and angiogenesis plays an important role in the process. Angiogenesis dysregulation is an important feature of malignant tumors. Studies suggest that, Survivin and a variety of human tumor angiogenesis has a close relationship. Glioma is the most common malignant tumors of the central nervous system, and its incidence is approximately 45% of intracranial tumors. Despite the comprehensive treatment, including surgery, radiotherapy and chemotherapy, including glioma has been improving, but the therapeutic effect of the disease has not yet been significantly improved median survival time of malignant glioma patients is still less than a year . Difficult to treat the root causes of glioma and its malignant characteristics are closely related. Studies have shown that the strong angiogenesis is one of the most important malignant phenotype of glioma. Reveal the key genes involved in glioma angiogenesis to overcome this chronic illness has important scientific significance and clinical value. At present, the role and mechanism of Survivin in glioma angiogenesis is not entirely clear, based on previous findings, we speculate that the formation of a Survivin-angiogenic factor between the mutually reinforcing positive feedback regulatory pathway in glioma thus to promote glioma angiogenesis, resulting in tumor progression. Based on this, the subject from the following three aspects. A pro-angiogenic factors Objective To investigate the pro-angiogenic factor VEGF, bFGF and PDGF glioma cell line SHG44 and U251 Survivin expression in glioma cells expression of Survivin. In cultured human glioma cell line SHG-44 (no expression of Survivin) and U251 (high expression of Survivin) were added to the culture medium of recombinant human VEGF, bFGF and PDGF, each cell is divided into a number of groups, respectively, containing The final concentration with 0, 25, 50, 100 ng / ml VEGF, 0,1,2.5,5,10,20 ng / ml bFGF and 0,5,10,20,40 ng / ml PDGF cultured in DMEM culture medium for 24 hours after collecting the total protein of the cells in each group; the same time, each cell containing a final concentration of 100ng/ml VEGF, 10ng/ml of bFGF, 10ng/ml PDGF culture broth culture 0,6,24,48,72 h , the same collection of total protein. By Western blot method to detect the above Survivin protein expression levels. To extract the total RNA of the cells in each group, with the former, each cell containing a final concentration of 100ng/ml VEGF, 10ng/ml bFGF, 10ng/ml PDGF culture broth culture 0,3,6,12,24 h . Real-time RT-PCR to detect the survivin gene RNA expression levels. PDGF raised the SHG-44 cells and U251 cells Survivin gene expression at the transcriptional level, and this raised the role of a concentration-and time-dependent. The SHG44 10ng/ml PDGF role 3h and 12H Survivin gene mRNA and protein expression levels reached a peak, then decreased gradually, minimum 5 ng / ml levels of PDGF role after 24h raised SHG44 cells Survivin protein expression levels. For the the U251, 10ng/ml PDGF role in the 6 ~ 12h and 24 to 48h, Survivin gene RNA and protein levels reached a peak, then gradually decreased, a minimum 5ng/ml of PDGF effect 24 hours after the same visible U251 cells Survivin protein expression levels up high. bFGF raised U251 cells Survivin gene expression at the transcriptional level, and presents a time-and concentration-dependent manner, while no effect on SHG-44 cells. 10ng/ml bFGF role in U251 cells 6 and 12 to 24 hours, the Survivin gene mRNA and protein levels, respectively, reached a peak, then gradually decreased below 10ng/ml bFGF after 24h no Survivin expression level. VEGF Survivin gene expression of two types of cells had no increase in design time and concentration within the role. Conclusion In addition to the direct stimulation of vascular endothelial cell proliferation, migration and play a pro-angiogenic effect, may also indirectly through Survivin gene-mediated signaling pathways involved in glioma blood vessel formation process of angiogenic factors bFGF and PDGF. The the Survivin gene eukaryotic expression vector and RNA interference vector construction, Identification and cells transfected with Objective To construct the the Survivin gene eukaryotic expression vector and Survivin gene-specific RNA interference vector, and transfected into human glioma cell line SHG44 U251, establish a stable transfected cell lines. Reverse transcriptase polymerase chain reaction amplification of the the Survivin gene encoding sequence, amplification product was double digested and cloned into the eukaryotic cell expression vector pcDNA3.1 was successfully constructed the the Survivin gene eukaryotic expression vector pcDNA3.1-SVV. Designed and synthesized for Survivin gene for firefly luciferase (FLF) gene-specific RNA interference clips, the latter as independent insertion sequence for human gene targeting, respectively, the two interference fragment ligated into vector was successfully constructed. Survivin gene RNA interference the carrier and unrelated sequences carrier. Liposome SHG44 cells transfected with pcDNA3.1, pcDNA3.1-SVV, pSilencer3.1-SVV, pSilencer3.1-FLF and the transfection to pSilencer3.1-H1neo of U251 cells stably transfected cell G418 screening system, using real-time RT-PCR, Western blot and immunocytochemistry were used to detect the expression of Survivin gene in transfected cells. Results of restriction enzyme digestion and nucleotide sequence analysis confirmed the successful build the Survivin gene eukaryotic expression vector pcDNA3.1-SVV and Survivin gene RNA interference vector pSilencer3.1-SVV; obtained stably transfected dye pcDNA3.1, pcDNA3.1 -SVV, pSilencer3.1-SVV, pSilencer3.1-FLF, and pSilencer3.1-H1neo the cell line, named SHG44-P, SHG44-S, U251-S, U251-F and U251-P. Identification of protein and RNA levels, SHG44-S cells Survivin gene stable expression of Survivin gene expression was significantly inhibited U251-S cells, the inhibition rate of 70%, SHG44-P cells Survivin gene expression, U251-F and U251 -P cells Survivin gene expression did not change significantly. Conclusion The eukaryotic expression vector pcDNA3.1-SVV make the Survivin gene expression in SHG44 cells in stable. Survivin gene-specific RNA interference vector pSilencer3.1-SVV able to significantly inhibit Survivin gene expression in U251 cells. The Survivin gene for glioma cells in blood vessel formation mechanism experimental basis. 3 pro-angiogenic factor stable transfection in vitro glioma cells express purpose of change to compare gene transfection SHG44 and U251 glioma cell lines before and after the three kinds of pro-angiogenic factor VEGF, bFGF and PDGF expression changes, explore whether the Survivin gene formed by the three pro-angiogenic factor involved in glioma-mediated angiogenesis. The method of in vitro SHG-44, SHG44-P, SHG44-S and U251, U251-P, U251-F and U251-S total cellular RNA and total protein were extracted. Real-time RT-PCR, Western blot, were used to detect three promote angiogenesis factor VEGF, bFGF and PDGF mRNA and protein expression in the cells in each group. Each group were collected cell culture supernatants by ELISA to detect the expression of three pro-angiogenic factor secretion. The experimental results of image and statistical analysis. Result, the level of protein, SHG44-S cytoplasmic and supernatant in the expression of bFGF and no VEGF the PDGF expression of, SHG44-P and SHG44 cytoplasmic supernatant never showed expression of VEGF, bFGF and PDGF; U251-S cytoplasmic and supernatant VEGF, bFGF expression level decreased significantly decreased before transfection level of 50% and 30%, no change in levels of PDGF expression. However, Blank and unrelated sequences carrier of transfection, U251-P and U251-F cells VEGF, bFGF and PDGF protein levels average no significant change. results are consistent with the results of mRNA level and protein level. Conclusion Survivin gene expression increase SHG44 cells the expression of bFGF from scratch; inhibition of survivin gene expression in U251 cells of VEGF, bFGF expression was significantly inhibited. Tip the Survivin gene form factor (VEGF) and basic fibroblast growth factor mediated through the blood vessels involved in glioma angiogenesis, this provides the basis for a regulatory pathway for further study of the Survivin gene involved in glioma angiogenesis.
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