Angiostatin(K1-3)和endostatin裸DNA联合治疗人脑胶质瘤的实验研

Aim In order to investigate the feasibility and characteristic of antiangiogenesis, the research of treatment of glioma were conducted. Method： Human angiostatin K(1-3) and endostatin gene fragments were cloned and sequenced by RT-PCR methods. Construction of angiostatin K(1-3) or endostatin eukaryotic expressed vector，which angiostatin K(1-3) and endostatin cDNA were linked with human Ig Kappa-chain secretive singal by PCR, the PCR product was cloned into eukaryotic expressed vector to construct pcDNA-SAK(1-3) or pcDNA-S- endostatin.The above eukaryotic expression vectors were transfected into human SHG44 glioma cell by lipofectamine method and the positive clones were selected by G418. The biological characters of glioma cells in experimental group, empty vector group and control group were examined by electron microscope, flow cytometry and cell growth rate counting, and there were no differences of cell cycles in these three groups. Immunohistochemistry, immunofluorescence,western blot and hybridization in situ confirmed that the protein of angiostatin K(1-3) and endostatin were expressed in the experimental group. The SHG44 cells were planted under the skin of nude mice to eastablish nude mice model and the tumorigenesis of the cells were observed and compared among these three groups.The nude DNA packaged by lipofactamine was injected to the tumor，the effects of angiostatin K(1-3) and endostatin protein to the nude mice were discussed. Results：Angiostatin K(1-3) and endostatin gene fragements were got and sequenced right. The pcDNA-SAK(1-3) or pcDNA-S-endostatin were constructed, which were correct by sequenceing and restriction enzyme analysis. The expressed angiostaitn K(1-3) or endostatin can inhibit chicken-embryo angiogenesis in vivo and didn't affect the biological activities of SHG44 gliomas cells. The tumorigenesis of experimental group cells compared with that of empty vector group or control group was greatly reduced. The tumor necrosis rate of experimental group was obviously higher than that of empty vector group or control group.The blood vessel numbers of experimental group were lower than those of empty vector group or control group. Conclusion：The above results illustrated that it is feasible to treat the human glioma under the skin of nude mice with the eukaryotic expressed angiostatin K(1-3) or endostatin nude cDNA. The tumor growth suppression in vivo was caused by angiostatin K(1-3) or endostatin due to its antiangiogenesis and tumor necrosis. Our experimental studies lay the preliminary foundation for further treatment of glioma and the other solid tumors by antiangiogenesis methods.

将构建在真核表达载体上的angiostatin(K1-3)和endostatin分别或联合注入荷脑胶柿雎闶蟾骨弧⒕猜龊土鎏迥冢τ妹庖咦榛⒃辉咏弧CR和Southern Blot检测其体内泶铮教致鉊NA注射后对体内器官的影响和抑瘤性，检测血液内各项生化指标的改变，为抗血管生成基因药物治疗人脑胶质瘤奠定理论和实践基础。这方面研究有重要临床应用价值。