【作者】 柯璐琳；

【导师】 许云禄；

【作者基本信息】 福建医科大学 ， 药理学， 2010， 硕士

【摘要】 解离素(disintegrin)是一类富含半胱氨酸的小分子多肽,其含有精-甘-天冬氨酸(RGD)序列或赖-甘-天冬氨酸(KGD)序列能抑制依赖此识别位点的整合素与其配体结合,从而抑制血小板聚集形成瘤栓、肿瘤血管新生,达到抑制肿瘤细胞侵润、迁移的目的。本文拟从江浙短尾蝮(Gloydius brevicaudus,GBV)蛇毒中分离纯化解离素GBV-Ⅳ4,并测定生物学活性,观察其抑制血管生成和肿瘤转移作用。为进一步开发利用提供实验依据。1.江浙短尾蝮蛇毒解离素的分离纯化根据福建医科大学蛇毒所分离纯化江浙短尾蝮蛇毒解离素的方法应用Superdex 75凝胶过滤色谱、DEAE Sepharose Fast Flow离子交换色谱和Lichrospher C18反相色谱等技术从江浙短尾蝮蛇毒中分离纯化解离素GBV-Ⅳ4。GBV-Ⅳ4在SDS-PAGE (Tris-Tricine系统)凝胶电泳中,呈单一蛋白条带。采用经典的Born法,证明其能抑制ADP诱导的血小板聚集。2. GBV--Ⅳ4对人脐静脉内皮细胞(HUVEC)小管形成的影响采用小管形成实验观察GBV-Ⅳ4对HUVEC的抗血管生成,发现GBV-Ⅳ4可剂量依赖地抑制HUVEC管腔形成。GBV-Ⅳ4 0.15、0.3和0.6mg/ml使小管形成数量分别由PBS组的35.667±6.062减少至15.556±4.925、9.556±3.046和2.222±1.563;小管形成面积分别由PBS组的2616.198±312.022(μm2)减少至1002.198±431(μm2)、421.621±52(μm2)和115.926±102.01(μm2);小管形成周长分别由PBS组的965.431±143.991(μm)减少至435.059±168.221(μm)、180.944±68.178(μm)和71.25±61.26(μm)。3. GBV-Ⅳ4对b-FGF诱导的小鼠体内基质胶血管生成的影响小鼠体内基质胶血管生成实验检测GBV-Ⅳ4对血管生成的影响。组织切片免疫组化染色后计数b-FGF阳性对照、b-FGF+GBV-Ⅳ4 1.25mg/kg、b-FGF+ GBV-Ⅳ4 2.5mg/kg和PBS阴性对照各组基质胶内微血管数。GBV-Ⅳ4可剂量依赖地抑制基质胶内微血管的形成,b-FGF组微血管数为17.13±3.20,b-FGF+GBV-Ⅳ4 1.25mg/kg、b-FGF+GBV-Ⅳ4 2.5mg/kg组分别为9.80±1.95和2 .33±0.75,PBS组为0.4±0.44。4. GBV-Ⅳ4对小鼠黑色素瘤细胞B16F1实验性肺转移的影响实验性肺转移实验检测GBV-Ⅳ4对B16F1体内转移的影响。GBV-Ⅳ4 1.25mg/kg和2.5mg/kg能显著减少转移瘤数,PBS、GBV-Ⅳ4 1.25mg/kg和2.5mg/kg作用下小鼠肺结节数分别为181.14±61.00、118.75±17.44和73.87±34.21。免疫组化染色显示GBV-Ⅳ4 1.25mg/kg和2.5mg/kg能显著减少肿瘤组织内血管的新生,PBS、GBV-Ⅳ4 1.25mg/kg和2.5mg/kg作用下肿瘤结节内微血管数分别为28.40±2.70、24.4±3.36和11.4±5.77。结论:用Superdex 75凝胶过滤色谱、DEAE Sepharose Fast Flow离子交换色谱和Lichrospher C18反相色谱从短尾蝮蛇毒粗毒中分离解离素是稳定、可行的技术路线。GBV-Ⅳ4能抑制ADP诱导的血小板聚集及具有较强的抗肿瘤血管生成作用。更多还原

【Abstract】 Disintegrin which contains the amino acid sequence Arg-Gly-Asp(RGD) or Lys-Gly-Asp(KGD),has been implicated as a recognition site in interactions between integrin and its ligands. It inhibits infiltration and migration of the malignant cell by anti-aggregation of platelet aggregation which formed tumor embolus and induced angiogenesis of tumor. This study was designed to isolate and purify disintegrin from Gloydius brevicaudus venom and study its characterization and anti-angiogenesis, anti-tumor metastasis activities.1.Isolation of disintegrin GBV-Ⅳ4 from the venom of Gloydius brevicaudusAccording to the protocol of isolation of disintegrin from Gloydius brevicaudus venom(GBV-) of Lab Snake Venom Research of Fujian Medical University ,we isolated disintegrin(GBV-Ⅳ4) from GBV- by Superdex 75 Gel filtration chromatography, DEAE Sepharose Fast Flow ion-exchange, and Lichrospher C18 reverse chromatography. It was homogeneous as a single band showed on SDS-polyacrylmide gel electrophoresis (SDS-PAGE, Tris-Tricine system) . Effects of GBV-Ⅳ4 on inhibition platelet aggregation induced by ADP were assayed by the Born’s method, .2. The effect of GBV-Ⅳ4 on human umbilical vein cells (HUVEC) tube-like structuresThe tube formation assay of HUVEC was used to study anti-angiogenesis of GBV-Ⅳ4. GBV-Ⅳ4 could significantly inhibit the tube formation of HUVEC in a dose-dependent manner. When HUVEC were treated with GBV-Ⅳ4 0.15mg/ml, 0.3mg/ml, 0.6mg/ml or PBS, the number of tube was 15.556±4.925, 9.556±3.046, 2.222±1.563 and 35.667±6.062 respectively. The area (μm2) of tube formation was 1002.198±431, 421.621±52, 118.926±34.003 and 2616.198±312.022 respectively. The perimeter (μm) of tube formation was 435.059±168.221, 180.944±68.178, 71.25±61.26 and 965.431±143.991 respectively.3. The effect of GBV-Ⅳ4 on anti-angiogenesis in the Matrigel plug in miceMatrigel plug assay was used to investigate anti-angiogenesis of GBV-Ⅳ4 in mice. GBV-Ⅳ4 could significantly inhibit microcapsule formation in the matrigel which was induced by b-FGF. When mice were treated with b-FGF, b-FGF + GBV-Ⅳ4 1.25mg/kg , b-FGF + GBV-Ⅳ4 2.5mg/kg or PBS, the number of microcapsule in the matrigel was 17.13±3.20、9.80±1.95 and 2 .33±0.75 and 0.4±0.44 respectively.. 4. The effect of GBV-Ⅳ4 on artificial lung metastasis of B16F1 in miceThe artificial lung metastasis assay was used to measure anti-metastasis of GBV-Ⅳ4. Mice were treated with GBV-Ⅳ4 of 1.25mg/kg, 2.5mg/kg or PBS after injection of B16F1. The metastatic nodes of B16F1 on lung surface were significantly reduced by the GBV-Ⅳ4. The number of metastatic nodes in GBV-Ⅳ4 1.25mg/kg, 2.5mg/kg or PBS treated mice was 118.75±17.44, 73.87±34.21 and 181.14±61.00 respectively. GBV-Ⅳ4 also significantly inhibits the microcapsule genesis in the metastatic node. The number of microcapsule in GBV-Ⅳ4 1.25mg/kg, 2.5mg/kg or PBS treated mice was 24.4±3.36, 11.4±5.77 and 28.40±2.70 77 respectively.ConclusionsDisintegrin (GBV-Ⅳ4 ) could be purified from Gloydius brevicaudus venom by Superdex 75 Gel filtration chromatography, DEAE Sepharose Fast Flow ion-exchange and Lichrospher C18 reverse chromatography. It was a potent platelet aggregation inhibitor and significantly inhibits angiogenesis and metastasis of tumor.更多还原