【作者】 周先丽；

【导师】 雷丹青；

【作者基本信息】 广西医科大学 ， 药理学， 2006， 硕士

【摘要】 目的从尖吻蝮蛇毒中分离纯化一种小分子多肽,研究其理化性质,对ADP、胶原、凝血酶诱导的血小板聚集以及对血管生成的影响。方法经Sephadex G-75凝胶过滤,超滤,DEAE-Sepharose CL-6B离子交换层析法分离纯化蛇毒组分,暂命名为K组分,采用高效液相鉴定纯度,用SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)测定其分子量,等电聚焦法测定等电点,用比浊法测定其抗血小板聚集活性,流式细胞术分析该组分与血小板GPⅡb/Ⅲa的作用,酶联免疫吸附实验测血浆中GMP-140含量,电镜下观察K组分对血小板超微结构的影响,建立鸡胚绒毛尿囊膜模型测定其抗血管生成活性。结果从尖吻蝮蛇毒中分离相对分子量约为7862D等电点为4.29的组分。紫外扫描发现,K组分在200-230nm处有最大吸收。该组分能抑制由ADP、胶原、凝血酶诱导的血小板聚集并成剂量依赖性;剂量依赖性抑制FITC - CD41a与血小板GPⅡb/Ⅲa的结合;能减轻血小板超微结构的损伤程度;具有抑制鸡胚尿囊膜血管生成的作用。结论此法成功地从尖吻蝮蛇毒中纯化出去整合素。该组分有很强的抗血小板聚集作用。更多还原

【Abstract】 AIM To purify a disintegrin from Agkistrodon Acutus snake venom , and to study the physical and chemical properties of the peptide, as well as its effects on the platelet aggregation stimulated by ADP, collagen, and thrombin. Method To extract snake venom through Superdex 75 gel filtration, ultrafiltration and DEAE -Sepharose CL-6B ionexchange. The purified product was identified by HPLC C18. The molecular weight was determined by SDS- polyacrylamid gel electrphoresis. The isoelectric point was estimated by the isoelectric focusing electrophoresis. Platelet aggragation was measured by nephelometry. The effect of fraction K on GPⅡb/Ⅲa was detected by flow eytometry. The level of GMP-140 in plasma was measured by ELISA. The ultrastructures of platelet were observed as well. The antiangiogenesis effect in vivo of fraction K on CAM assay was observed. Result The molecular weight of a small peptide purified from Agkistrodon Acutus snake venom was 7862D. Its isoelectric point was pH4.29. This peptide could dose-dependently inhibit the platelet aggregation induced by ADP, collagen and thrombin,and the binding reaction of fluorescein isothiocyanate (FITC)-conjugated CD41a.The peptide could lighten the disruption of platelets ultrastructure and inhibit the spontaneous angiogenesis of CAM. And it had no effect on GMP-140 in plasma. Conclusion The method has been proved to be successful for the purification of disintegrin that inhibits platelet aggregation.更多还原