地鳖虫蛋白提取物的体内抗肿瘤作用研究
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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。
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.
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。
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.
引文
[1] 武忠弼．病理学[M]．第四版．北京：人民卫生出版社，1996．125，130，136～137，154～162．
[2] 姜文奇，张晓实，朱孝峰，李志铭．肿瘤生物治疗学[M]．广州：广东科技出版社，2006．1，45，47，87，96．
[3] 尚玮，钟启平．细菌制剂抗肿瘤及免疫调节作用的研究进展[J]．天津医科大学学报，2006，12(3)：474～477．
[4] Pfeffer LM, Dinarello CA, Herberman RB, et al. Biological properties of recombinant alpha-interferons: 40th anniversary of the discovery of interferons [J]. Cancer Res, 1998,58(12):2489～2499.
[5] 汤钊猷．现代肿瘤学[M]．上海：上海医科大学出版社，1993．71～72，81～83．
[6] 田志刚，孙汭．肿瘤治疗第四种模式——生物疗法[J]．实用肿瘤学杂志，1991，5(3)：76～79．
[7] 李文军 等．恶性肿瘤的生物治疗进展[J]．人民军医，1996，7：55～58．
[8] 陆志城．贝伐单抗：一种治疗转移性结直肠癌的新药[J]．中国处方药，2005，38(5)：32～34．
[9] 路力生 等．抗癌效应细胞与肿瘤生物治疗[J]．实用肿瘤杂志，1990，5(4)：196～199．
[10] Steven A．Rosenberg．癌症生物治疗——原理与实践[M]．陈复兴 等 主译．北京：人民军医出版社，2005．884～886．
[11] Gilboa E, Nair SK, Lyerly HK. Immunotherapy of cancer with dendritic-cell-based vaccines [J]. Cancer Immunol Immunother, 1998,46(2): 82～87.
[12] Cai DW, Mukhopadhyay T, Liu Y, Fujiwara T, Roth JA. Stable expression of wild-type p53 gene in human lung cancer cells after retrovirus-mediated gene transfer [J]. Hum Gene Ther, 1993,4: 617.
[13] Johnson JR, Bross P, Cohen M, et al. Approval summary: imatinib mesylate capsules for treatment of adult patients with newly diagnosed philadelphia chromosome-positive chronic myelogenous leukemia in chronic phase [J]. Clin Cancer Res, 2003,9:1972～1979.
[14] Herbst RS, Giaccone G, et al. Gefitinib in combination with paclitaxel and carboplatin in advanced non-small-cell lung cancer: a phase Ⅲ trial-INTACT 2 [J]. J Clin Oncol, 2004,22:785～794.
[15] 梁斌，宋旭红，黄东阳．维甲酸衍生物对肿瘤细胞的凋亡诱导作用[J]．中国药理学通报，2006，22(3)：257～262．
[16] Algire GH, Chalkley HW, Legallais FY, Park HD. Vascular reactions of normal and malignant tissues in vivo. I. Vascular reactions of mice to wounds and to normal and neoplastic transplants [J]. J Natl Cancer Inst, 1945: 73～85.
[17] Folkman. Tumor angiogenesis : therapeutic implications [J]. N Engl J Med, 1971,285: 1182～1186.
[18] Weidner N, Folkman J, Pozza F, et al. Tumor angiogenesis: new significant and independent prognostic indicator in earlystage breast carcinoma [J]. J Natl Cancer Inst,1992,84: 1875～1887.
[19] Lu C, Tanigawa N. Spontaneous apoptosis is inversely related to intratumoral microvessel density in gastric carcinoma [J]. Cancer Res, 1997,57:221～224.
[20] Bergers G, Javaherian R, Lo KM, Folkman J, Hanahan D. Effects of angiogenesis inhibitors on multistage carcinogenesis in mice [J]. Science, 1999,284:808～812.
[21] Boehm T, Folkman J, Browder T, O'Reilly MS. Antiangiogenic therapy of experimental cancer does not induce acquired drug resistance [J]. Nature, 1997,390:404～407.
[22] O'Reilly MS, Holmgren L, Shing Y, et al. Angiostatin: a novel angiogenesis inhibitor that mediates the suppression of metastases by a Lewis lung carcinoma [J]. Cell, 1994,79:315～328.
[23] O'Reilly MS, Boehm T, Shing Y, et al. Endostatin: an endogenous inhibitor of angiogenesis and tumor growth [J]. Cell, 1997, 88: 277～285.
[24] 丁怡 等．雷公腾甲素对血管内皮细胞尿激酶型纤溶酶原激活物表达的影响[J]．癌症，2005，24(7)：801～805．
[25] 张前 等．羟基红花黄色素A抑制新生血管形成的机制研究[J]．北京中医药大学学报，2004，27(3)：25～29．
[26] 杨军 等．新生霉素抑制血管生成及其与长春新碱的协同作用[J]．药学学报，2003，38(10)：731～734．
[27] 张维东 等．蝎毒多肽提取物抗肿瘤血管生成作用的实验研究[J]．中国药理学通报，2005，21(6)：708～711．
[28] 陈鑫 等．三氧化而砷对人乳腺癌裸鼠抑制肿瘤血管生成的作用[J]．重庆医学，2003，32(6)：708～709．
[29] 逯海燕 等．氧化吲哚类化合物的体内抑瘤活性及其血管生成抑制作用[J]．中国药理学与毒理学杂志，2003，17(6)：401～407．
[30] Kohn EC, Liotta LA. L651582: a novel antipriliferative and anti-metastasis agent [J]. J Natl Cancer Inst, 1990,82:54～60.
[31] 杜钢军 等．反应停的抗新生血管形成及抗肿瘤作用研究[J]．中国药理学通报，2005，21(4)：471～474．
[32] Kohn EC, Alessandro R, Spoonster J, Wersto RP, Liotta LA. Angiogenesis: role of calcium-mediated signal transduction [J]. Pro Natl Acad Sci USA, 1995,92: 1307～1311.
[33] Kohn EC, Figg WD, Sarosy GA, et al. Phase Ⅰ trial of micronized formulation carboxyamido-triazole, in patients with refractory solid tumors: pharmacokinetics, Clinical outcome, and comparison of formulations [J]. J Clin Oncol, 1997,15: 1985～1993.
[34] Mellin GW, Katsenstein M. The saga of thalidomide [J]. N Engl J Med,1962,264: 1184～1193.
[35] Moreira AL, Sampaio EP, Zmuidzinas A, Frindt P, Smith KA, Kaplan G. Thalidomide exerts its inhibitory action on tumor necrosis factor alpha by enhancing mRNA degradation [J]. J Exp Med, 1993,177: 1675～1680.
[36] Or R, Feferman R, Shoshan S. Thalidomide reduces vascular density in granulation tissue of subcutaneously implanted polyvinyl alcohol sponge in guinea pigs [J]. Exp Hematol, 1998,26: 217～221.
[37] Primrose JN, Bleiberg H, Daniel F, et al. Marimastat in recurrent colorectal cancer: exploratory evaluation of biological activity by measurement of carcinoembryonic antigen [J]. Br J Cancer, 1999, 79: 509～514.
[38] Price A, Shi Q, Morris D, et al. Marked inhibition of tumor growth in a glioma tumor model by a novel synthetic matrix metalloproteinase inhibitor AG3340 [J]. Clin Cancer Res, 1999,5: 845～854.
[39] Fong TA, Shawyer LK, Sun L, et al. SU-5416 is potent and selective inhibitor of the vascular endothelial growth factor receptor (Flk-I/KDR) that inhibits tyrosine kinase catalysis, tumor vascularization, and growth of multiple tumor types [J]. Cancer Res, 1999,59: 99～106.
[40] Gutheil JC, Campbell TN, Pierce PR, et al. Phase I study of vitaxin, an anti-angiogenic humanized monoclonal antibody to vascular integrin avβ3 [J]. ProcAm Soc Clin Oncol, 1998,17: 215a(abst).
[41] Hurmitz HI, Fehrenbacher L, Hainsworth JD, et al. Bevacizumab in combination with fluorouracil and leucovorin: an active regimen for first-line metastatic colorectal cancer [J]. J Clin Oncol, 2005, 23: 3502～3508.
[42] Heidenreich R, Machein M, Nicolaus A, et al. Inhibition of solid tumor growth by gene transfer of VEGF receptor-1 mutants [J]. Int J Cancer, 2004, 111(3):348～357.
[43] Tanaka T, Cao Y, Folkman J, Fine HA. Viral vector-targeted antiangiogenic gene therapy utilizing an angiostatin complementary DNA [J]. Cancer Res, 1998,58: 3362～3369.
[44] 黄金保 等．土鳖虫抗兔心脑缺氧实验研究[J]．长治医学院学报，1994，1：102～104．
[45] 陈瑛 等．水蛭、土鳖虫水体物对高分子葡聚糖所致大鼠血瘀模型的纤溶系统的影响[J]．中药药理与临床，1995，1：33．
[46] 杨耀芳 等．土鳖虫对血虚小鼠红细胞免疫功能的实验研究[J]．中国免疫学杂志，2003，19(10)：686～689．
[47] 郭庆民 等．土鳖虫对红细胞变形性和膜成分的影响[J]．中国生化药物杂志，2000，21(5)：235～237．
[48] 于燕 等．土鳖虫水提物对实验性高血脂血症大鼠内皮和内皮素的影响[J]．中国生化药物杂志，2003，24(1)：15～17．
[49] 韩雅莉，李张伟．地鳖虫纤溶活性蛋白的纯化及性质研究[J]．生物工程学报，2006，22(4)：639～643．
[50] 韩雅莉，谢昆．土鳖虫糖蛋白的提取及抗肿瘤活性初步研究[J]．汕头大学学报(自然科学版)，2006，21(4)：46～50．
[51] 尹鸣 等．紫杉醇抗血管生成作用的实验研究[J]．肿瘤防治研究，2004，31(5)：282～285．
[52] 张永雨 等．龙须菜藻胆蛋白抗突变与抗肿瘤作用的研究[J]．中国海洋药物杂志，2005，24(3)：36～38．
[53] 林厚文 等．海兔中新环氧甾醇的体外抗肿瘤活性研究[J]．第二军医大学学报，2004，25(5)：479～481．
[54] 冼励坚，潘启超．蛋白激酶C调节剂及一个有希望的新药——苔藓虫素(Bryostatin)[J]．中国癌症杂志，1996，6(1)：121～125．
[55] 杨振淮 等．蟾酥注射液对小鼠S180和人结肠癌HT-29裸鼠移植性肿瘤的影响[J]．中华肠胃外科杂志，2005，8(2)：169～171．
[56] 夏明钰 等．厚朴酚通过改变Bax／Bcl-X_L表达和激活caspase诱导人宫颈癌HeLa细胞凋亡[J]．中国药理学通报，2005，21(3)：318～322．
[57] 齐晓娟 等．雷公藤对荷S180小鼠的抑瘤作用的实验研究[J]．齐齐哈尔医学院学报，2005，26(2)： 124～125．
[58] 孙文平 等．中药川莲治疗小鼠肉瘤S180的实验研究[J]．大连医科大学学报，1997，19(2)：88～90．
[59] 商澎 等．当归多糖AP-0对小鼠移植性肿瘤的抑制作用[J]．第三军医大学学报，23(11)：1299～1302．
[60] 国家药典委员会．中华人民共和国药典：2000年版．一部．北京：化学工业出版社，2000．16．
[61] 韩雅莉，李张伟．地鳖虫纤溶成分的分离纯化和活性测定[J]．中药材，2006，29(8)：765～767．
[62] 王维澎．大黄蛰虫丸应用三则[J]．北京中医，2000，5：51．
[63] 徐叔云，卞如濂，陈修．药理实验方法学[M]．第三版．北京：人民卫生出版社，2003．431，434～435，535，1819～1820．
[64] 张尔贤，俞丽君．鼠尾藻多糖清除氧自由基作用的研究Ⅱ——UVc鼠尾藻对多糖抗氧化作用的影响[J]．中国海洋药物，1997，16(3)：1～4．
[65] 李建武 等．生物化学实验原理和方法[M]．北京：北京大学出版社，1994．313～317．
[66] 陈惠黎．生物化学检验技术[M]．北京：人民卫生出版社，1990．240～241．
[67] 蒲丹 等．重组冠状病毒核衣壳蛋白血清抗体检测[J]．免疫学杂志，2004，20(5)：397～399．
[68] Gavtieli Y, et al. Identification of programmed cell death in suit specific labeling of nuclear DNA fragmentation [J]. J Cell Biol, 1992,119:493～500.
[69] Peitsch MC, et al. DNA fragmentation during apoptosis is caused by frequent single-strand cuts [J]. Nucle Acids Res, 1993,18:4206～4209.
[70] 金艳书 等．苦参碱对人肝癌细胞增殖、细胞周期及细胞凋亡的影响[J]．中国临床康复，2006，10(3)：107～109．
[71] Folkman J, Haundenschild CC, Zetter BR. Long-term culture of capillary endothelial cells [J]. Proc Natl Acad Sci USA, 1979,76: 5217～5221.
[72] 成惠林．检测细胞活性的MTT方法[J]．江苏医学，1996，22(5)：330～331．
[73] 司徒镇强，吴军正．细胞培养[M]．修订版．西安：世界图书出版西安公司，2004．251～252．
[74] 张维东 等．蝎毒多肽提取物的抗血管生成作用[J]．细胞生物学杂志，2005，27(3)：343～346．
[75] 赵卫红，寿好长 等．细胞凋亡[M]．河南：河南医科大学出版社，1997．256～180．
[76] 袁兰，陈英玉，张立．用激光扫描共聚焦显微镜原位检测细胞凋亡[J]．现代仪器，2003，1：47～49．
[77] Fairbairn DW, Olive PL, O' Neill KL. The comet assay:A comprehensive review[J]. Mutat Res, 1995,339: 37～59.
[78] Singh NP, McCoy MT, Tice RR. A simple technique for quantitation of low levels of DNA damage in individual cells [J]. Exp Cell Res, 1988,175(1): 184～191.
[79] Auerbach R, Kubai L, Knighton D, Folkman J. A simple procedure for the long-term cultivation of chicken embryos [J]. Dev Biol, 1974,41: 391.
[80] Ribatti D, De-Falco G, Nico B, et al. In vivo time-course of the angiogenic response induced by multiple myeloma plasma cells in the chick embryo chorioallantoic membrane [J]. J Anat, 2003, 203(3): 323～328.
[81] 李晓岑．冬虫夏草对肝硬化大鼠谷丙转氨酶和谷草转氨酶的影响[J]．中国林副特产，2005，4：24．
[82] 于善谦．免疫学导论[M]．北京：高等教育出版社，1999．64，252．
[83] 张志荣 等．靶向治疗分子基础与靶向药物设计[M]．北京：科学出版社，2005．251
[84] 刘超英 等．血管生长因子与肿瘤血管生成[J]．肿瘤防治杂志，2001，8(5)：560～562．
[85] 李家增．恶性肿瘤与止血障碍[J]．癌症进展杂志，2005，3(2)：110～113．
[86] 王学锋，王鸿利．恶性肿瘤与血栓形成[J]．诊断学理论与实践，2002，1(2)：66～69．
[87] 张琳 等．肝素类化合物与肿瘤的关系[J]．食品与药品，2005，7(4)：13～16．
[88] 张培彤 等．活血药对人肺癌细胞粘附和侵袭的影响[J]．中国中西医结合杂志，1999，19(2)：103～105．
[89] 凌桂琴，王绪．COX-2与肿瘤fJ]．徐州医学院学报，2005，25(3)：271～274．
[90] 韩锐．抗癌药物与实验技术[M]．北京：北京医科大学中国协和医科大学联合出版社，1997．316～319，349～352，368～376．
[91] 林春姬 等．低剂量博来霉素A_5促进荷瘤小鼠化疗效果及其机制探讨[J]．吉林大学学报(医学版)，2004，30(3)：354～356．
[92] 吴琛珩 等．恶性肿瘤的氧化还原态及其抗氧化能力的改变[J]．中国病理生理杂志，2005，21(11)：2160～2165．
[93] Folkman J, Klagsburn M. Angiogenic factors [J]. Science, 1987,235: 442～447.
[94] Senger DR, Galli SJ, Dvorak AM, Perruzzi CA, Harvey VS. Tumor cells secrete a vascular permeability factor that promotes accumulation of ascites fluid [J]. Science, 1983,219: 983～985.
[95] Folkman J. What is the evidence that tumors are angiogenesis dependent? [J]. J Natl Cancer Inst, 1990,82:4～6.
[96] Nguyen M, Shing Y, Folkman J. Quantitation of angiogenesis and anti-angiogenesis in the chick embryo chorioallantoic membrane [J]. Microvasc Res, 1994,47: 31～40.
[97] 仉文升，李安良．药物化学[M]．第二版．北京：高等教育出版社，2005．286．
[2] 姜文奇，张晓实，朱孝峰，李志铭．肿瘤生物治疗学[M]．广州：广东科技出版社，2006．1，45，47，87，96．
[3] 尚玮，钟启平．细菌制剂抗肿瘤及免疫调节作用的研究进展[J]．天津医科大学学报，2006，12(3)：474～477．
[4] Pfeffer LM, Dinarello CA, Herberman RB, et al. Biological properties of recombinant alpha-interferons: 40th anniversary of the discovery of interferons [J]. Cancer Res, 1998,58(12):2489～2499.
[5] 汤钊猷．现代肿瘤学[M]．上海：上海医科大学出版社，1993．71～72，81～83．
[6] 田志刚，孙汭．肿瘤治疗第四种模式——生物疗法[J]．实用肿瘤学杂志，1991，5(3)：76～79．
[7] 李文军 等．恶性肿瘤的生物治疗进展[J]．人民军医，1996，7：55～58．
[8] 陆志城．贝伐单抗：一种治疗转移性结直肠癌的新药[J]．中国处方药，2005，38(5)：32～34．
[9] 路力生 等．抗癌效应细胞与肿瘤生物治疗[J]．实用肿瘤杂志，1990，5(4)：196～199．
[10] Steven A．Rosenberg．癌症生物治疗——原理与实践[M]．陈复兴 等 主译．北京：人民军医出版社，2005．884～886．
[11] Gilboa E, Nair SK, Lyerly HK. Immunotherapy of cancer with dendritic-cell-based vaccines [J]. Cancer Immunol Immunother, 1998,46(2): 82～87.
[12] Cai DW, Mukhopadhyay T, Liu Y, Fujiwara T, Roth JA. Stable expression of wild-type p53 gene in human lung cancer cells after retrovirus-mediated gene transfer [J]. Hum Gene Ther, 1993,4: 617.
[13] Johnson JR, Bross P, Cohen M, et al. Approval summary: imatinib mesylate capsules for treatment of adult patients with newly diagnosed philadelphia chromosome-positive chronic myelogenous leukemia in chronic phase [J]. Clin Cancer Res, 2003,9:1972～1979.
[14] Herbst RS, Giaccone G, et al. Gefitinib in combination with paclitaxel and carboplatin in advanced non-small-cell lung cancer: a phase Ⅲ trial-INTACT 2 [J]. J Clin Oncol, 2004,22:785～794.
[15] 梁斌，宋旭红，黄东阳．维甲酸衍生物对肿瘤细胞的凋亡诱导作用[J]．中国药理学通报，2006，22(3)：257～262．
[16] Algire GH, Chalkley HW, Legallais FY, Park HD. Vascular reactions of normal and malignant tissues in vivo. I. Vascular reactions of mice to wounds and to normal and neoplastic transplants [J]. J Natl Cancer Inst, 1945: 73～85.
[17] Folkman. Tumor angiogenesis : therapeutic implications [J]. N Engl J Med, 1971,285: 1182～1186.
[18] Weidner N, Folkman J, Pozza F, et al. Tumor angiogenesis: new significant and independent prognostic indicator in earlystage breast carcinoma [J]. J Natl Cancer Inst,1992,84: 1875～1887.
[19] Lu C, Tanigawa N. Spontaneous apoptosis is inversely related to intratumoral microvessel density in gastric carcinoma [J]. Cancer Res, 1997,57:221～224.
[20] Bergers G, Javaherian R, Lo KM, Folkman J, Hanahan D. Effects of angiogenesis inhibitors on multistage carcinogenesis in mice [J]. Science, 1999,284:808～812.
[21] Boehm T, Folkman J, Browder T, O'Reilly MS. Antiangiogenic therapy of experimental cancer does not induce acquired drug resistance [J]. Nature, 1997,390:404～407.
[22] O'Reilly MS, Holmgren L, Shing Y, et al. Angiostatin: a novel angiogenesis inhibitor that mediates the suppression of metastases by a Lewis lung carcinoma [J]. Cell, 1994,79:315～328.
[23] O'Reilly MS, Boehm T, Shing Y, et al. Endostatin: an endogenous inhibitor of angiogenesis and tumor growth [J]. Cell, 1997, 88: 277～285.
[24] 丁怡 等．雷公腾甲素对血管内皮细胞尿激酶型纤溶酶原激活物表达的影响[J]．癌症，2005，24(7)：801～805．
[25] 张前 等．羟基红花黄色素A抑制新生血管形成的机制研究[J]．北京中医药大学学报，2004，27(3)：25～29．
[26] 杨军 等．新生霉素抑制血管生成及其与长春新碱的协同作用[J]．药学学报，2003，38(10)：731～734．
[27] 张维东 等．蝎毒多肽提取物抗肿瘤血管生成作用的实验研究[J]．中国药理学通报，2005，21(6)：708～711．
[28] 陈鑫 等．三氧化而砷对人乳腺癌裸鼠抑制肿瘤血管生成的作用[J]．重庆医学，2003，32(6)：708～709．
[29] 逯海燕 等．氧化吲哚类化合物的体内抑瘤活性及其血管生成抑制作用[J]．中国药理学与毒理学杂志，2003，17(6)：401～407．
[30] Kohn EC, Liotta LA. L651582: a novel antipriliferative and anti-metastasis agent [J]. J Natl Cancer Inst, 1990,82:54～60.
[31] 杜钢军 等．反应停的抗新生血管形成及抗肿瘤作用研究[J]．中国药理学通报，2005，21(4)：471～474．
[32] Kohn EC, Alessandro R, Spoonster J, Wersto RP, Liotta LA. Angiogenesis: role of calcium-mediated signal transduction [J]. Pro Natl Acad Sci USA, 1995,92: 1307～1311.
[33] Kohn EC, Figg WD, Sarosy GA, et al. Phase Ⅰ trial of micronized formulation carboxyamido-triazole, in patients with refractory solid tumors: pharmacokinetics, Clinical outcome, and comparison of formulations [J]. J Clin Oncol, 1997,15: 1985～1993.
[34] Mellin GW, Katsenstein M. The saga of thalidomide [J]. N Engl J Med,1962,264: 1184～1193.
[35] Moreira AL, Sampaio EP, Zmuidzinas A, Frindt P, Smith KA, Kaplan G. Thalidomide exerts its inhibitory action on tumor necrosis factor alpha by enhancing mRNA degradation [J]. J Exp Med, 1993,177: 1675～1680.
[36] Or R, Feferman R, Shoshan S. Thalidomide reduces vascular density in granulation tissue of subcutaneously implanted polyvinyl alcohol sponge in guinea pigs [J]. Exp Hematol, 1998,26: 217～221.
[37] Primrose JN, Bleiberg H, Daniel F, et al. Marimastat in recurrent colorectal cancer: exploratory evaluation of biological activity by measurement of carcinoembryonic antigen [J]. Br J Cancer, 1999, 79: 509～514.
[38] Price A, Shi Q, Morris D, et al. Marked inhibition of tumor growth in a glioma tumor model by a novel synthetic matrix metalloproteinase inhibitor AG3340 [J]. Clin Cancer Res, 1999,5: 845～854.
[39] Fong TA, Shawyer LK, Sun L, et al. SU-5416 is potent and selective inhibitor of the vascular endothelial growth factor receptor (Flk-I/KDR) that inhibits tyrosine kinase catalysis, tumor vascularization, and growth of multiple tumor types [J]. Cancer Res, 1999,59: 99～106.
[40] Gutheil JC, Campbell TN, Pierce PR, et al. Phase I study of vitaxin, an anti-angiogenic humanized monoclonal antibody to vascular integrin avβ3 [J]. ProcAm Soc Clin Oncol, 1998,17: 215a(abst).
[41] Hurmitz HI, Fehrenbacher L, Hainsworth JD, et al. Bevacizumab in combination with fluorouracil and leucovorin: an active regimen for first-line metastatic colorectal cancer [J]. J Clin Oncol, 2005, 23: 3502～3508.
[42] Heidenreich R, Machein M, Nicolaus A, et al. Inhibition of solid tumor growth by gene transfer of VEGF receptor-1 mutants [J]. Int J Cancer, 2004, 111(3):348～357.
[43] Tanaka T, Cao Y, Folkman J, Fine HA. Viral vector-targeted antiangiogenic gene therapy utilizing an angiostatin complementary DNA [J]. Cancer Res, 1998,58: 3362～3369.
[44] 黄金保 等．土鳖虫抗兔心脑缺氧实验研究[J]．长治医学院学报，1994，1：102～104．
[45] 陈瑛 等．水蛭、土鳖虫水体物对高分子葡聚糖所致大鼠血瘀模型的纤溶系统的影响[J]．中药药理与临床，1995，1：33．
[46] 杨耀芳 等．土鳖虫对血虚小鼠红细胞免疫功能的实验研究[J]．中国免疫学杂志，2003，19(10)：686～689．
[47] 郭庆民 等．土鳖虫对红细胞变形性和膜成分的影响[J]．中国生化药物杂志，2000，21(5)：235～237．
[48] 于燕 等．土鳖虫水提物对实验性高血脂血症大鼠内皮和内皮素的影响[J]．中国生化药物杂志，2003，24(1)：15～17．
[49] 韩雅莉，李张伟．地鳖虫纤溶活性蛋白的纯化及性质研究[J]．生物工程学报，2006，22(4)：639～643．
[50] 韩雅莉，谢昆．土鳖虫糖蛋白的提取及抗肿瘤活性初步研究[J]．汕头大学学报(自然科学版)，2006，21(4)：46～50．
[51] 尹鸣 等．紫杉醇抗血管生成作用的实验研究[J]．肿瘤防治研究，2004，31(5)：282～285．
[52] 张永雨 等．龙须菜藻胆蛋白抗突变与抗肿瘤作用的研究[J]．中国海洋药物杂志，2005，24(3)：36～38．
[53] 林厚文 等．海兔中新环氧甾醇的体外抗肿瘤活性研究[J]．第二军医大学学报，2004，25(5)：479～481．
[54] 冼励坚，潘启超．蛋白激酶C调节剂及一个有希望的新药——苔藓虫素(Bryostatin)[J]．中国癌症杂志，1996，6(1)：121～125．
[55] 杨振淮 等．蟾酥注射液对小鼠S180和人结肠癌HT-29裸鼠移植性肿瘤的影响[J]．中华肠胃外科杂志，2005，8(2)：169～171．
[56] 夏明钰 等．厚朴酚通过改变Bax／Bcl-X_L表达和激活caspase诱导人宫颈癌HeLa细胞凋亡[J]．中国药理学通报，2005，21(3)：318～322．
[57] 齐晓娟 等．雷公藤对荷S180小鼠的抑瘤作用的实验研究[J]．齐齐哈尔医学院学报，2005，26(2)： 124～125．
[58] 孙文平 等．中药川莲治疗小鼠肉瘤S180的实验研究[J]．大连医科大学学报，1997，19(2)：88～90．
[59] 商澎 等．当归多糖AP-0对小鼠移植性肿瘤的抑制作用[J]．第三军医大学学报，23(11)：1299～1302．
[60] 国家药典委员会．中华人民共和国药典：2000年版．一部．北京：化学工业出版社，2000．16．
[61] 韩雅莉，李张伟．地鳖虫纤溶成分的分离纯化和活性测定[J]．中药材，2006，29(8)：765～767．
[62] 王维澎．大黄蛰虫丸应用三则[J]．北京中医，2000，5：51．
[63] 徐叔云，卞如濂，陈修．药理实验方法学[M]．第三版．北京：人民卫生出版社，2003．431，434～435，535，1819～1820．
[64] 张尔贤，俞丽君．鼠尾藻多糖清除氧自由基作用的研究Ⅱ——UVc鼠尾藻对多糖抗氧化作用的影响[J]．中国海洋药物，1997，16(3)：1～4．
[65] 李建武 等．生物化学实验原理和方法[M]．北京：北京大学出版社，1994．313～317．
[66] 陈惠黎．生物化学检验技术[M]．北京：人民卫生出版社，1990．240～241．
[67] 蒲丹 等．重组冠状病毒核衣壳蛋白血清抗体检测[J]．免疫学杂志，2004，20(5)：397～399．
[68] Gavtieli Y, et al. Identification of programmed cell death in suit specific labeling of nuclear DNA fragmentation [J]. J Cell Biol, 1992,119:493～500.
[69] Peitsch MC, et al. DNA fragmentation during apoptosis is caused by frequent single-strand cuts [J]. Nucle Acids Res, 1993,18:4206～4209.
[70] 金艳书 等．苦参碱对人肝癌细胞增殖、细胞周期及细胞凋亡的影响[J]．中国临床康复，2006，10(3)：107～109．
[71] Folkman J, Haundenschild CC, Zetter BR. Long-term culture of capillary endothelial cells [J]. Proc Natl Acad Sci USA, 1979,76: 5217～5221.
[72] 成惠林．检测细胞活性的MTT方法[J]．江苏医学，1996，22(5)：330～331．
[73] 司徒镇强，吴军正．细胞培养[M]．修订版．西安：世界图书出版西安公司，2004．251～252．
[74] 张维东 等．蝎毒多肽提取物的抗血管生成作用[J]．细胞生物学杂志，2005，27(3)：343～346．
[75] 赵卫红，寿好长 等．细胞凋亡[M]．河南：河南医科大学出版社，1997．256～180．
[76] 袁兰，陈英玉，张立．用激光扫描共聚焦显微镜原位检测细胞凋亡[J]．现代仪器，2003，1：47～49．
[77] Fairbairn DW, Olive PL, O' Neill KL. The comet assay:A comprehensive review[J]. Mutat Res, 1995,339: 37～59.
[78] Singh NP, McCoy MT, Tice RR. A simple technique for quantitation of low levels of DNA damage in individual cells [J]. Exp Cell Res, 1988,175(1): 184～191.
[79] Auerbach R, Kubai L, Knighton D, Folkman J. A simple procedure for the long-term cultivation of chicken embryos [J]. Dev Biol, 1974,41: 391.
[80] Ribatti D, De-Falco G, Nico B, et al. In vivo time-course of the angiogenic response induced by multiple myeloma plasma cells in the chick embryo chorioallantoic membrane [J]. J Anat, 2003, 203(3): 323～328.
[81] 李晓岑．冬虫夏草对肝硬化大鼠谷丙转氨酶和谷草转氨酶的影响[J]．中国林副特产，2005，4：24．
[82] 于善谦．免疫学导论[M]．北京：高等教育出版社，1999．64，252．
[83] 张志荣 等．靶向治疗分子基础与靶向药物设计[M]．北京：科学出版社，2005．251
[84] 刘超英 等．血管生长因子与肿瘤血管生成[J]．肿瘤防治杂志，2001，8(5)：560～562．
[85] 李家增．恶性肿瘤与止血障碍[J]．癌症进展杂志，2005，3(2)：110～113．
[86] 王学锋，王鸿利．恶性肿瘤与血栓形成[J]．诊断学理论与实践，2002，1(2)：66～69．
[87] 张琳 等．肝素类化合物与肿瘤的关系[J]．食品与药品，2005，7(4)：13～16．
[88] 张培彤 等．活血药对人肺癌细胞粘附和侵袭的影响[J]．中国中西医结合杂志，1999，19(2)：103～105．
[89] 凌桂琴，王绪．COX-2与肿瘤fJ]．徐州医学院学报，2005，25(3)：271～274．
[90] 韩锐．抗癌药物与实验技术[M]．北京：北京医科大学中国协和医科大学联合出版社，1997．316～319，349～352，368～376．
[91] 林春姬 等．低剂量博来霉素A_5促进荷瘤小鼠化疗效果及其机制探讨[J]．吉林大学学报(医学版)，2004，30(3)：354～356．
[92] 吴琛珩 等．恶性肿瘤的氧化还原态及其抗氧化能力的改变[J]．中国病理生理杂志，2005，21(11)：2160～2165．
[93] Folkman J, Klagsburn M. Angiogenic factors [J]. Science, 1987,235: 442～447.
[94] Senger DR, Galli SJ, Dvorak AM, Perruzzi CA, Harvey VS. Tumor cells secrete a vascular permeability factor that promotes accumulation of ascites fluid [J]. Science, 1983,219: 983～985.
[95] Folkman J. What is the evidence that tumors are angiogenesis dependent? [J]. J Natl Cancer Inst, 1990,82:4～6.
[96] Nguyen M, Shing Y, Folkman J. Quantitation of angiogenesis and anti-angiogenesis in the chick embryo chorioallantoic membrane [J]. Microvasc Res, 1994,47: 31～40.
[97] 仉文升，李安良．药物化学[M]．第二版．北京：高等教育出版社，2005．286．