【作者】 许云禄；

【导师】 陈晓春；

【作者基本信息】 福建医科大学 ， 内科学， 2008， 博士

【摘要】 1.短尾蝮蛇毒解离素的分离纯化及理化性质测定应用Superdex 75和Sephadex G-25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中纯化得一个解离素纯品,暂定名为GbvⅣ4。GbvⅣ4在SDS-PAGE(Tris-Tricine系统)凝胶电泳中,呈单一蛋白条带,质谱法测定其相对分子质量为7442Da,采用聚丙烯酰胺凝胶盘状电泳测定其等电点约为6.3。自动电位滴定法测定磷脂酶A2活力和Rick方法测定蛋白酶活力,结果均为0。氨基酸序列测定发现它由70个氨基酸组成,含有RGD三肽活性中心,6对二硫键,符合中链解离素特征。通过Genbank检索,未发现与GbvⅣ4序列完全一致的氨基酸序列,是一种新的解离素蛋白。2. GbvⅣ4对血小板聚集的影响采用经典的Born法,发现GbvⅣ4在体外可抑制多种聚集剂诱导的血小板聚集,且该作用呈明显的量效关系,GbvⅣ4对ADP、凝血酶诱导的血小板聚集的EC50分别为0.298μg/ml和0.577μg/ml。3. GbvⅣ4对人脐静脉内皮细胞株ECV304生长的影响以SRB法观察GbvⅣ4对人脐静脉内皮细胞株ECV304生长的抑制作用,测定其IC50,结果GbvⅣ4使细胞突起回缩,细胞变圆,细胞浮起不再贴壁且聚集成簇,失去正常的铺路石状。GbvⅣ4呈剂量依赖性的抑制ECV304的生长,其作用24h的IC50为25.42μg/ml。4. GbvⅣ4对鸡胚尿囊膜(Chick Chorioallantoic membrane,CAM)新生血管生成的影响选用鸡胚尿囊膜模型观察GbvⅣ4对鸡胚尿囊膜血管生成的抑制作用,采用测量区血管面积与测量区面积的比值(VA/CAM)评价抑制程度。结果显示的GbvⅣ4 3.098和11.79μg处理后可见鸡胚CAM血管生成减少,分布稀疏,分支减少,但形态基本正常,呈树状分布,与NS组相比VA/CAM显著减少(P﹤0.01)。5. GbvⅣ4对黑色素瘤B16细胞体外粘附能力的影响细胞-基质粘附实验观察GbvⅣ4对B16细胞粘附能力的影响。GbvⅣ4使细胞在给药后4h细胞突起回缩变圆,且聚集成团簇状。采用MTT法,发现GbvⅣ4可抑制肿瘤细胞与纤维连接蛋白的粘附作用,抑制作用呈浓度依赖性,在终浓度16μg/ml时抑制率为59.34%。6. GbvⅣ4对黑色素瘤B16细胞体外迁移能力的影响采用划痕实验检测B16细胞体外迁移能力。在划痕后48h,对照组的细胞在划痕处已经基本长满,而GbvⅣ4组的无细胞区仍较明显。7. GbvⅣ4对黑色素瘤B16和B16–F10细胞体外侵袭的影响应用Transwell小室重建基底膜侵袭实验观察GbvⅣ4对B16和B16–F10细胞侵袭能力的影响,药物与细胞作用48h后,发现GbvⅣ4对B16和B16–F10细胞穿越基底膜的能力显著降低,穿过基底膜的细胞数量与GbvⅣ4的浓度成反比.8. GbvⅣ4对黑色素瘤B16–F10小鼠实验性肺转移模型的影响采用裸BALB/c小鼠黑色素瘤B16–F10实验转移模型观察解离素对肿瘤转移的影响,解离素腹腔注射明显减少接种黑色素瘤B16–F10细胞小鼠的肺重和肺转移灶数量,解离素(50、100μg/kg×14 d)和环磷酰胺(50 mg/kg×2 d)对B16黑色素瘤细胞肺转移的抑制率分别为67.8%、82.8%和48.1%。结论:从江浙短尾蝮蛇毒中分离得到一个解离素纯品GbvⅣ4,由70个氨基酸组成,含有RGD三肽活性中心,分子量为7442Da,等电点6.3。GbvⅣ4能剂量依赖地抑制ADP及凝血酶诱导的血小板聚集;对人脐静脉内皮细胞株ECV304的生长有明显的抑制作用;GbvⅣ4具有抑制鸡胚尿囊膜血管生成的作用;GbvⅣ4在体外能明显降低肿瘤细胞的粘附、迁移、侵袭能力。对小鼠B16黑色素瘤的实验性转移肺癌有显著的抑制作用。更多还原

【Abstract】 Disintegrin which originate of snake and leech venom ,contains the amino acid sequence Arg-Gly-Asp(RGD) or Lys-Gly-Asp(KGD). RGD is the common sequence which exists in extracellular matrix(ECM),as fibrinogen、fibronectin、thrombospondin,osteopontin and von willebrand factor(vWF) etc, has been implicated as a recognition site in interactions between integrin and its ligands.It exhibit a wide array of biological activities, such as inhibit platelet aggregation, angiogenesis and tumor migration.This study was designed to isolate and purify a disintegrin from Gloydius brevicaudus venom and analyse its characterization and biological activities.1.Purification and characterization of the disintegrin from Gloydius brevicaudus Venom(Gbv)Seven fractions(Ⅰ～Ⅶ) were isolated from Gbv by Gel filtration chromatography on Superdex 75 colum . The fractionⅣinhibited the platelet aggregations induced by ADP with molecular weight lower than 10000 dalton,so it suggested as a contained disintegrin component. A disintegrin named GbvⅣ4 was purified from the fraction by Sephadex G-25 gel filtration, 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) with molecular weight 8746 dalton as calculated by Image Master VDS system.The isoelectric point of GbvⅣ4 was pI 6.3 by disc polyacrylamide gel eletrophoresis. GbvⅣ4 exhibited no detectable phospholipase A2(PLA2)activity with the pH-stat technique and proteinase activity according to the method of Rick. GbvⅣ4 is composed of 70 amino acid sequence with RGD active region and molecular weight 7442 dalton as assayed by Mass Spectrography. Characteration of GbvⅣ4 is consistent with meta-chain disintegrin’s(70 amino acid sequence, six pairs of disulfide bond). Retrieved by Genbank , GbvⅣ4 has high homology with other disintegrins .Above all,we conclude that GbvⅣ4 is a new kind of disintegrn.2. The effects of GbvⅣ4 on the human platelet aggregation.According to the Born’s method, the effects of GbvⅣ4 on platelet aggregation induced by ADP and thrombin were assayed. The results showed that GbvⅣ4 could dose-dependently inhibite ADP- and thrombin-induced platelet aggregation with IC50 0.339 and 0.577μg/ml respectively.3.The effect of GbvⅣ4 on the proliferation of human umbilical vein ECV304 cellsThe inhibition of GbvⅣ4 on cultured ECV304 cells was observed and IC50 value was assayed by SRB method.The result showed that GbvⅣ4 inhibited the proliferation of ECV304 cells in a dose-dependent manner with IC50(24h) 25.42μg/ml. Furthermore,The ECV304 cells undergo remarkable morphological change by treatment with GbvⅣ4:the cells retracted ,turn to ground, formed gaps and multicelluar aggregation.4.The effect of GbvⅣ4 on angiogenesis of chick chorioallantoic membrane(CAM)The antiangiogenesis effect in vivo of GbvⅣ4 on CAM assay was observed and the the ratio of area of blood vessel and area of CAM in measure area (VA/CAM) was used to asses the inhibition.The result showed that GbvⅣ4(3.098、11.79μg)inhibited the angiogenesis in vivo: the morphology of blood vessels on CAM were basically normal,but the blood vessels distributed scarcely .The VA/CAM was less than control group significantly.5. The effects of GbvⅣ4 on B16 cells’adhesive capability to fibronection (Fn)Cell-matrix adhesion assay was used to study the adhesive ability of B16 cells.With phase microscope observation we found the B16 cells looked like epidermoid cells and were polygonal in shape with the typical cobblestone growing pattern, but the B16 cells treated with GbvⅣ4 undergo remarkable morphological change :the cells retracted ,turn to ground、formed gaps and multicelluar aggregation. According to MTT method, different concentration of GbvⅣ4 showed inhibitory effects, and the inhibited effects was dose dependent, the inhibition ratio was 59.34% at the concentration of 16μg/ml.6. The effects of GbvⅣ4 on B16 cells’migration on Fibronection.The migration of B16 cells was observed by erasion trace test.The results showed that GbvⅣ4 could inhibit B16 cells from moving on Fibronection effectively.7. The effects of GbvⅣ4 on B16 and B16-F10 cells’invasion on MatrigelTranswell chamber assay was applied to measure the effect of GbvⅣ4 on invasion of B16 and B16-F10 cells. After 48 h, the invasion of B16 and B16-F10 cells was less than those in control groups, and the number of invasive cells was dose dependent.8. Inhibitory effects of GbvIV on metastases of mouse B16 melanomaThe effects of GbvIV on metatasis were studied by using an experimental metastasis model of mouse BI6-F10 melanoma cell line In mice that received i.v.injection of mouse B 16-F10 melalloma cells,daily i.p.GbvIV remakably reduced lung weight, and number of lung metastases.Lung metastasis inhibition rates of GbvIV(50、100μg/ml) and cyclophosphamide(50 mg/kg) on B16 -F10 melanoma cells were 67.8% ,82.8% ,and 48.1% ,respectively.ConclusionsA disintegrin GbvⅣ4 was purified from Gloydius brevicaudus venom with molecular weight 7442 dalton and pI 6.3.It is composed of 70 amino acid sequence with RGD active region . It was a potent platelet aggregation inhibitor. GbvⅣ4 inhibited proliferation of human umbilical vein endothelial cells and antiangiogenesis of CAM. GbvⅣ4 has strong activity of anti-tumor cell adhesion、migration、invasion and metastases.更多还原