【作者】 郭桅；

【导师】 韩雅莉；

【作者基本信息】 汕头大学 ， 生物化学与分子生物学， 2007， 硕士

【摘要】 地鳖虫是《中华人民共和国药典》收录的活血化瘀类动物中药材。目前研究发现该虫含有丰富的纤溶活性蛋白组分，在体内外均显示出明显的抗血栓效果。地鳖虫入药能明显提高机体的纤溶能力，改善恶性肿瘤患者体内的高凝状态，降低血栓并发症，进而缓解癌症症状。民间有将地鳖虫入药治疗多种恶性肿瘤的例子，但尚未见到此类研究的相关文献报道。应用现代药理学方法对地鳖虫的抗肿瘤活性成分及其作用机理进行系列研究十分必要。本文通过生化途径，提取纯化了地鳖虫纤溶活性蛋白组分，研究了该蛋白提取物的体内抑瘤效果，发现了该纤溶活性蛋白能抑制新生血管生成，为进一步研究地鳖虫蛋白提取物的抗肿瘤机理提供了切入点。本研究中，用不同浓度的地鳖虫蛋白提取物(0.2～0.8g／ml，生药量)作用于S180肉瘤荷瘤小鼠，观察各组的抑瘤效果及相关生理指标的差异；同时分别用地鳖虫蛋白粗提物和经盐析、离子交层析以及分子筛层析等技术对地鳖虫蛋白粗提物进一步纯化得到的活性蛋白，作用于鸡胚尿囊膜，观察它们对新生血管生成的抑制作用。研究结果显示，地鳖虫蛋白粗提物对S180肉瘤荷瘤小鼠有显著的抑瘤作用；蛋白粗提物和纯化的活性蛋白对鸡胚尿囊膜新生血管的生成均有明显抑制作用，其中纯化品的抑制活性显著高于粗品，以生药量计算显示一定的量效关系；且二者对尿囊膜下的鸡胚胎生长发育的影响较阳性对照(地塞米松组)小，差异显著。由此显示，地鳖虫蛋白提取物有较好的体内抑瘤作用及血管生成抑制活性。另外，本研究通过建立实体型肝癌H22荷瘤小鼠模型，分别以地鳖虫蛋白提取物对其进行连续灌胃(ig.)、隔2日腹腔注射(ip.)等处理，发现实体瘤的生长被明显抑制。分析各实验组小鼠肝组织MDA含量、SOD活力、GPT水平、GOT水平和血清GPT水平等生化指标，结果显示地鳖虫蛋白处理组动物的肝组织抗氧化能力明显增强，其生理状态也优于模型对照组；其次，发现地鳖虫蛋白处理组动物的肿瘤组织SOD活力明显高于模型对照组，显示地鳖虫蛋白提取物在提高机体纤溶能力的同时，也改变了实体瘤组织生长的生化状态；对实验小鼠血清抗H22肿瘤抗体水平的检测，发现阳性药物对照组抗体水平显著降低，而地鳖虫蛋白处理组则明显升高，这显示了地鳖虫蛋白提取物对实体瘤生长的抑制机理不同于阳性对照药环磷酰胺，环磷酰胺能直接杀伤肿瘤细胞，但对免疫系统的抑制则是重要的副作用之一，地鳖虫蛋白对抗H22肿瘤抗体的水平的提高，可能是其抑瘤机理的一部分。由于地鳖虫纤溶活性蛋白显示了良好的器官水平新生血管抑制作用，而在新生血管生成过程中血管内皮细胞起着十分重要的作用。通过体外细胞培养实验，发现提取的地鳖虫纤溶活性蛋白能够抑制人微血管内皮细胞(MVEC)的增殖；另外Annexin V-FITC／PI双荧光染色检测、单细胞凝胶电泳(SCGE)检测结果显示，地鳖虫纤溶活性蛋白能诱导MVEC凋亡。地鳖虫作为一种传统的活血化瘀类动物药材，能明显提高机体的纤溶状态，改善血液循环。通过本项研究进一步表明，其纤溶活性蛋白提取物对实体瘤有明显的抑制作用，且能显著改善荷瘤动物的生理状态。此外，它还呈现出显著的新生血管抑制活性，这提示它极可能是通过血管生成抑制途径产生体内抑瘤效应的。然而，它抑制肿瘤血管增生的具体途径及其机制也尚需进一步研究加以揭示。更多还原

【Abstract】 Eupolyphaga sinensis Walker collected in CP (2000 edition) as a kind of Chinese TraditionalMedcine is commonly used as anticoagulant. The recent studies find that Eupolyphaga sinensisWalker contains protein groups with fibrinolytic activity, which will enhance the level ofanti-thrombus in human body. It is also used in several kinds of cancers, because it can relievethrombus symptom during the developing tumors. But its anti-tumor activity reported on theacademic journals is not exist yet. Thus studies on its anti-tumor activity and the relatedmechanism with new pharmacological techniques should be urgently taken. At the begining ofthis study, we acquire the protein groups with fibrinolytic activity from fresh Eupolyphagasinensis Walker through a series of biochemical methods, and the next research found that theacquired protein groups could inhibit the mice’s tumor growth in vivo and suppress angiogenesisthrough CAM(Chick Embryo Chorioallantoic Membrane) assay. Then the results would be afootstone for further researches.During the study, the mice with S180 sarcoma inoculated were treated(i.g) with theprotein extractions from Eupolyphaga sinensis Walker at three different doses (0.2 g/ml, 0.4g/ml, 0.8 g/ml, as crude materials), and the inhibitory rates of tumor growth has beencalculated seven days later. We found that tumors from experimental groups treated with theprotein extractions were suppressed remarkably. CAM assay has been used to determine theneovascularizative effect in vivo with the crude proteins and the extractions purified byammonium sulphate precipitation, DEAE-cellulose and SephadexG-75 Columnchromatography. The protein extractions exhibited potent activity of anti-angiogenesis. Ourdata suggest that the proteins extracted from Eupolyphaga sinensis with fibrinolytic activityshould suppress the developing of cancers and inhibit angiogenesis respectively in vivo. Thechicken embryos treated with Eupolyphaga sinensis Walker protein have not been affected onappearance. But dexamethasone make the embryos maldeveloping as the positive control,considering its side effects.During another animal model in vivo, the mice inoculated with H22 liver cancer weretreated with crude proteins(i.g) and purified extractions(i.p) respectively. Then the inhibitoryrates of tumor growth turned out that the cancers have been suppressed remarkably in the experimental groups treated with Eupolyphaga sinensis Walker. At the same time, MDA,SOD, GPT, GOT level in liver, GPT level in serum, have been analyzed. Then the datasuggest mice inoculated with H22 liver cancer should be in a good physical condition treatedwith Eupolyphaga sinensis Walker protein, while the anti-oxidation capacities of their livershave been enhanced. The SOD activity in tumor tissues was different yet, which groupstreated with Eupolyphaga sinensis Walker protein contained much higher SOD activities thanthe control. It seems that protein with fibrinolytic activity should change the biochemicalconditions in tumor tissues. The levels of anti-H22 antibody in serum from the experimentalgroups were much higher than the control. However, the level of the antibody fromCyclophosphamide treated group was much lower than the control. Cyclophosphamide is akind of tumor cell toxin, which inhibits the normal cells derived from marrow also, includingB cells that could secrete antibodies.Effect of enhancing the tumor antibodies should be one ofthe anti-tumor pathways by Eupolyphaga sinensis Walker.The protein groups extracted from Eupolyphaga sinensis Walker with fibrinolytic activityinhibit angiogenesis on CAM, while the endothelial cells are very important on angiogenesisduring tumor growth. Experiments turned out that the protein groups with fibrinolyticactivity could inhibit the proliferation of endothelial cells and induce their apoptosis byAnnexin V-FITC/PI. and SCGE assay in vitro.The Eupolyphaga sinensis walker has been clinically used in thrombus symptom asChinese Traditional Medcine for centuries. This study find that the protein groups acquiredwith fibrinolytic activity exhibits potent anti-cancer activity in vivo, which is also consideredan angiogenic inhibitor by CAM assay in vivo and endothelial cell experiments in vitro.Finally, the detailed mechanism of its anti-angiogenic pathway during tumor growth wouldbe opened out after further researches.更多还原