青鲨软骨血管生成抑制因子的制备【毕业论文】.doc

本科毕业论文 （20 届） 青鲨软骨血管生成抑制因子的制备 专业：药学 目 录 摘要 1 Abstract 2 1前言 3 2实验材料与仪器 2.1实验材料 3 2.2主要试剂 2.3仪器 4 3实验方法 3.1分离提取总蛋白 3.2 (NH4)2SO4分级沉淀 3.3抗氧化活性测定 3.4鸡胚绒毛尿囊膜实验 3.5离子交换层析 3.6电泳实验 3.7 Sephadex G-75凝胶过滤层析 4实验 9 4.1(NH4)2SO4分级沉淀结果 9 10 4.3鸡胚绒毛尿囊膜实验结果 10 4.4离子交换层析 11 4.5电泳实验 12 4.6 Sephadex G-75凝胶过滤层析 13 5实验 15 5.1硫酸铵分级沉淀 15 5.2柱层析方法 15 5.3抗氧化活性研究 16 5.4新生血管生成抑制因子 16 参考文献 16 致谢 18  [摘要] 目的：软骨鱼软骨具有的血管生成抑制作用，。从青鲨软骨中分离纯化出具有较强抗氧化活性的蛋白，抑制新生血管的能力。方法：青鲨软骨PBS浸提，低温离心(NH4)2SO4分级沉淀。通过离子交换层析（IEC）和凝胶层析（GFC）进行分离纯化，SDS电泳检测蛋白分子量。DPPH清除率、羟自由基清除率、还原力三种体外抗氧化实验的抗氧化。结果：SDS电泳结果所示，考马斯亮蓝染色显示为单一条带，证明达到电泳纯，根据标准蛋白质的迁移率，测得其分子量为95.9 kDa。运用CAM）活性抑制模型进行药理学研究分析发现，8 μg BSFN）对新生血管的抑制率已经高达80.9%。实验表明它能显著抑制CAM膜新生血管生成并且抑制效果具有一定的浓度依赖性。DPPH清除率、羟自由基清除率、还原力三种体外抗氧化实验显示BSFN在10 mg/mL时DPPH清除率、羟自由基清除率、还原力分别为82.68%、78.15%、0.398结论：软骨资源高值化利用开辟一条新的途径。青鲨；血管抑制因子；抗氧化 The Preparation of angiogenesis inhibitor in Blue shark Cartilage Abstract: Objective: Preparation and activity evaluation of angiogenesis inhibitor from Blue shark Cartilage. Method: Break the organization of Blue shark Cartilage, and taking it as raw materials, and we can extract it with PBS. Centrifuging, we can get the crude protein by Precipitating (NH4)2SO4. In this way, we could obtain activitive protein components and Monomer by separating and puring through IEC and GFC. Evaluate the molecular weight with SDSelectrophoresis. Make use of ·DPPH clearance, ·OH clearance and reducing power in vitro to evaluate the antioxidant activity of sub parts and monomer. Detecting the inhibition of neovascularization of the Cartilage protein extracts, sub parts and the monomer with CAM. Result: The purified BSFN was homogeneous as a single band on a coomasie brilliant blue-stained 12 % SDSgel electrophresis. According to the migration rate of standard protein, the molecular weights of BSFN is 95.9 kDa. With CAM activity inhibition model, the inhibition rate of BSFN was up to 80.9% at the concentration of 8μg. Moreover, the dosages of the angiogenesis in