| Objective: Gene therapy is well recognized with the development of tumor molecular biology and gene engineering. Anti-angiogenesis is one of the promising methods. Glioma is characterized by an exceptionally high vascularization. So it can be studied as a model for the process of angiogenesis and anti-angiogenic therapy. Tumors must establish an adequate vascular network to acquire the nutrition necessary for growth and metastasis. Thus, avascular tumors that do not access a blood supply rarely grow beyond a few cubic millimeters. The aim of anti-angiogenesis is to target the tumor blood supply.O'Reilly et al first identified the antiangiogenic factor angiostatin, representing a fragment of plasminogen. It can efficiently inhibit the growth of a broad array of murine tumors established in mice, and is nontoxic such that tumors can be subjected to repeated treatment cycles, without exhibiting acquired resistance to therapy. Its tumor-suppressor activity may arise from its ability toinhibit the proliferation of endothelial cells by binding to the α/β -subunits of ATP synthase, by inducing apoptotic cell death, by subverting adhesion plaque formation and thereby inhibiting the migration and tube formation of endothelial cells, and /or by down-regulating VEGF expression. However, antiangiogenic therapy with angiostatin requires prolonged administration of recombinant protein in vivo, which is problematic as the production and purification of angiostatin have proven to be difficult. In an alternative way, gene therapy with plasmid encoding angiostatin may be effective.Many tumors contain hypoxic microenvironment, which has been associated with malignant progression, poor prognosis, metastasis and resistance to radiotherapy and chemotherapy. How they sense hypoxia, and respond by activating hypoxia-inducible genes to establish a blood system is central to cancer biology, the answers to which will provide new strategies for cancer treatment. The discovery of hypoxia-inducible factor-1 (HIF-1) gave an insight into the regulation of hypoxia-inducible genes. HIF-1, which is formed by the assembly of HIF-1α and HIF-1β during hypoxia, binds hypoxia-response elements (HREs) in enhancers of genes encoding angiogenic factors such as VEGF, and glycolysis-related proteins such as glycolytic enzymes, andglucose transporters 1 and 3 (Glut-1 and 3). HIF-loc is over-expressed in many tumor types compared with respective normal tissues, Whereas HIF-1 P is constitutively expressed, HIF-la is rapidly degraded during normoxia and dramatically stabilized and activated following hypoxia, by an oxygen-sensing and signaling process that remains an enigma. Downstream effectors of HIF-1, in particular VEGF, have been intensively investigated as targets for the treatment of solid vascular tumors, with some degree of success. Thus inhibition of VEGF expression in tumor xenografts leads to vascular regression, and decreased tumor growth. We reasoned that the simplest means of inhibiting HIF-1 effectors responsible for tumor growth, would be to target HIF-1 itself.In this paper, we established glioma by injection of C6 cells into the subcutaneous region of nude mice to observe the pathological properties of subcutaneous glioma. We used angiostatin and antisense HIF-1 a (aHIF) recombinant plasmid mediated by liposome to treat gliomas formed in nude mice in order to explore the expression of angiostatin and HIF-1 a in glioma, the influence on angiogenesis, apoptosis of tumor cells, and the suppression of tumor growth.Methods: Angiostatin and aHIF recombinant plasmid pcDNA were constructed by PCR. Then angiostatin and aHIFrecombinant plasmid were transferred to E.coli JM1O. After amplification, plasmids were extracted and digested with BamHI and Xho I . The DNA bands were observed in DNA agarose gel electrophoresis. C6 glioma cell was cultured in the RMPI1640 culturing fluid containing 10% calf blood serum in the 37°C, 5%C02, saturation humidity incubating box. The generation was spreaded in 2 to 3 days. Cell morphological properties were observed by inverted microscope, Giemsa' s colouration, H-E colouration. C6 cells (1 X106/100u 1) were subcutaneous implanted into 4 to 6 week old BALb/c nude mice to establish the tumor model. Mice were sacrificed when the diameter of tumors reached 5mm. H-E colouration was performed to see the morphologic properties of tumor. Gliomas were achieved in 33 nude mice. When the diameter of tumor reached 4mm, three groups were divided randomly: control, As and aHIF. Purified plasmids were diluted in a solution of 5% glucose in 0.01% Triton X-100, and mixed in a ratio of 3:1 (wt:wt) with DOTAP cationic liposomes, which is an efficient transfection vehicle. Final plasmid concentration was lmg/ml. Tumors reached 0. 4 cm (30~35mm3) in diameter after approximately 6-9 days, and were injected with 100 ul expression plasmid (100 ug). Tumor growth was determined by measuring two perpendicular diameters every three days. Tumor volume was calculated by formula V= n /6XLW2.Immunohistochemistry was performed to detect the expression of As and HIF-1 a protein. CD31 staining and TUNEL (terminal deoxynucleiotidyl transferase-mediated deoxyuridine triphophate-digoxigenin nick end labelling) staining were performed to assess the vascularity and to detect the apoptotic cells in situ.Results: Subcutaneous gliomas were achieved in all nude mice. Tumor appeared about 4 days after C6 cells injection and it grew as time went on. The tumor was nodular and grey, even invaded the muscle tissue. Necrosis appeared in the central part of the tumor. Glioma was highly cellular. Rounded cells and multinucleate giants cells were the main appearance. There were occasional mitoses. New blood vessels were scattered with endothelial cells swelling and thrombosis. Angiostatin and aHIF could delay the growth of tumor. The growth curve showed that tumor treated with angiostatin and aHIF grew slower than the control group. Tumor volume was 1519. 88 + 256. 54mm3 for the control group 15 days after gene therapy, whereas 1148. 03± 191. 51mm3 for the angiostatin group and 718.81±122. 24mm3 for the aHIF group. The difference between control group and angiostatin was statistical significant, so did the difference between control group and aHIF group. Immunohistochemitry showed the expression of angiostatin in gene therapy group increasedcompared to control group. And aHIF decreased the expression of HIF-1a protein. Vascular density in both angiostatin and aHIF gene therapy group decreased, and number of apoptotic cells increased. The difference of vascular density and apoptotic index (AI) between control group and As was statistical significant. So did the difference between control group and aHIF group.Conclusion: subcutaneous glioma in nude mice is easily achieved. And morphological study shows that it can be used for gene therapy. DOTAP cationic liposome is an efficient transfection vehicle. Angiostatin is more expressed after gene therapy. And aHIF can decrease the expression of HIF-1 a . Both angiostatin and aHIF can inhibit the angiogenesis, increase the apoptosis of tumor cells and delay the growth of subcutaneous glioma in nude mice.Significance: Glioma is the most common type among primary tumors in central nervous system with high mortality and poor prognosis, and it comprise 40-45% of all human brain tumors. Current comprehensive treatment, such as surgery, radiotherapy and chemotherapy, only provide short-term management of glioma. The medium survival time of patients with malignant glioma is less than 1 year. Tumors must establish an adequate vascular network to acquire the nutrition necessary for growth and metastasis. Angiostatin,...
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