【作者】 吴秋玲；

【导师】 孔庆志；

【作者基本信息】 湖北中医药大学 ， 中医内科学， 2011， 博士

【摘要】 肿瘤尤其是恶性肿瘤日益成为危害人类健康的主要杀手,是人类最主要的死亡原因之一。而恶性肿瘤的患者,绝大多数死于肿瘤的侵袭和转移。转移是导致临床治疗失败,影响肿瘤患者长期生存的主要因素。如何阻断肿瘤的侵袭和转移,是防治肿瘤的根本所在。自从“肿瘤生长依赖血管生成”的假说提出之后,肿瘤血管生长的调节机制和抗血管生成的研究得到广泛关注。断绝或减少肿瘤血管供应、抑制肿瘤血管生成是控制肿瘤生长和转移的重要途径之一。中药成分复杂多样,对肿瘤血管生成的影响可能是多靶点、多层次的。寻找新的高效低毒血管生成抑制剂,尤其从中药中筛选出可以抑制新生血管、阻断肿瘤转移途径的新药,已成为当前国内外学者关心的重要课题。我们的研究选择了虫类药物中抗肿瘤作用明显的水蛭和斑蝥为切入点进行研究。目的探讨中药水蛭、斑蝥对鸡胚绒毛尿囊膜(CAM)血管生成的影响；水蛭、斑蝥对人脐静脉血管内皮细胞(HUVEC)的影响；水蛭、斑蝥对小鼠移植肉瘤(S180)血管形成的影响。从血管生长因子(VEGF)、基质金属蛋白酶(MMP)和微血管密度(MVD)等角度出发研究抑制血管生长的机理,从抗血管生成方面进一步探讨水蛭、斑蝥抗肿瘤的作用机制,为临床中药治疗恶性肿瘤,防止肿瘤的侵袭和转移提供实验依据,为祖国医学治疗恶性肿瘤开辟新的途径。方法应用CAM技术、免疫组化技术等,观察水蛭、斑蝥的含药血清对CAM血管形成以及对体外培养血管内皮细胞的影响,观察水蛭、斑蝥对小鼠移植肿瘤的影响。实验分在体实验研究和离体实验研究两部分：1.在体实验研究建立鸡胚绒毛尿囊膜模型,观察水蛭、斑蝥含药血清对鸡胚尿囊膜微血管生长(血管数目和血管面积)的影响；进行水蛭、斑蝥水煎浓缩液对小鼠移植肿瘤的抗转移观察,观察S180腹水瘤小鼠瘤体重量、肿瘤抑制率等。用免疫组化的方法测定肿瘤组织金属基质蛋白酶(MMP)的表达、肿瘤组织血管内皮生长因子(VEGF)的表达、微血管密度(MVD)等指标。2.离体实验研究观察水蛭、斑蝥含药血清对体外脐静脉血管内皮细胞(HUVEC)增殖的影响。显微镜下观察细胞的形态学改变,并采用MTT法在酶联免疫检测仪上测定光吸收值(OD值),测定含药血清对HUVEC活力的影响。结果1.发育6日的鸡胚绒毛尿囊膜血管模型较适用于研究药物对血管生成的影响。水蛭、斑蝥对鸡胚绒毛尿囊膜新生血管数量有明显的抑制作用(P<0.01),对于血管面积也有一定的抑制作用。水蛭高剂量组与空白对照组、血清组相比较,有显著的抑制新血管生成的能力(P<0.01),水蛭低剂量组与空白对照组相比有一定作用但不显著((P=0.0814)；斑蝥高低剂量组与空白对照组和血清组相比较,抑制新生血管数的能力较强,有显著性差异(P<0.01)；空白对照组和血清组之间没有差异(P=0.8530)。水蛭高低剂量组有一定抑制血管面积的作用,但差异不具有显著性(P=0.4008,0.4173,0.7647,0.8449)；斑蝥高低剂量组有抑制血管面积的作用,其中高剂量组与空白对照组、血清组比较,具有显著性差异((P=0.0015,0.0006)；低剂量组有一定抑制血管面积的作用但不具有显著性差异(P=0.1325,0.1192)。空白对照组和血清组之间比较没有显著性差异(P=0.9003)。水蛭和斑蝥的高剂量组相比有差异,斑蝥的抑制作用更明显(P=0.0065)。2.与对照组比较,水蛭高低剂量组和斑蝥高低剂量组对小鼠移植肿瘤均有明显的抑制作用,瘤重明显减轻,(P=0.0118,0.0013,0.0001,0.0002)；水蛭高低剂量和斑蝥高低剂量组的抑瘤率与对照组相比,均为P<0.01。水蛭、斑蝥对S180瘤体微血管密度(MVD)的影响显示：斑蝥高低剂量组、水蛭高剂量组微血管数目减少较明显(P=0.0252,P=0.0024和P=0.0483)。免疫组化结果显示,VEGF蛋白在S180肉瘤组织中呈高表达,各用药组对VEGF的表达有不同程度的抑制作用,斑蝥高低剂量组和水蛭高剂量组对VEGF的表达有较明显的抑制作用(P=0.0145,0.0420,0.0023)。实验结果还显示对照组MMP-9呈高表达,而水蛭、斑蝥高低剂量组MMP-9呈低表达,具有显著性差异(P=0.0011,0.0016,0.0224,0.0078)。3.与对照组相比较,水蛭、斑蝥含药血清各组对体外脐静脉血管内皮细胞(HUVEC)的增殖具有一定的抑制作用。MTT法测定含药血清对HUVEC增殖作用的影响结果显示：斑蝥高、中、低剂量组吸光度(OD值)与对照组比较有显著性差异(P=5.7706E-09,0.003534,2.3148E-06),且随着浓度的升高其抑制作用更加明显,呈很好的剂量依赖关系；水蛭高中剂量组吸光度(OD值)与对照组比较有显著性差异(P=1.2854E-05,0.025)。细胞形态学观察结果：未经处理的正常对照组细胞形态正常,细胞呈梭形状,完整,未见凋亡细胞。而斑蝥高、中、低剂量组细胞体积皱缩变小,甚至细胞破裂或死亡,细胞密度明显下降。水蛭高中剂量组亦可见明显的细胞体积缩小,甚至细胞破裂或死亡,细胞密度明显下降。结论1.水蛭、斑蝥对鸡胚绒毛尿囊膜新生血管有一定的抑制作用。2.水蛭、斑蝥可抑制小鼠S180肉瘤的生长,降低S180肉瘤组织中血管内皮生成因子(VEGF)、基质金属蛋白酶9(MMP-9)的表达,降低S180肉瘤组织的微血管密度(MVD)。3.水蛭、斑蝥可一定程度抑制脐静脉血管内皮细胞(HUVEC)的增殖。虫类中药水蛭、斑蝥对肿瘤血管生成有一定的抑制作用,其抑制肿瘤作用的机理之一是抑制血管的生成。其机制可能是通过降低VEGF、MMP-9的表达,降低肿瘤组织的微血管密度(MVD)以及抑制血管内皮细胞的增殖来实现的。这为中药抑制血管生成以及抗肿瘤研究,奠定了一定的理论和实验基础。更多还原

【Abstract】 Malignant tumor is one of diseases seriously threatening mankind health and one of major causes of death. The deaths of most patients with malignant tumor result from the invasion and aversion of tumor, which are primary factors leading to failure of clinical treatment and relating to survival period of patients. Inhibition of invasion and aversion of tumor is the key point for precaution and treatment of tumor. As the hypothesis of tumor growth depending on angiogenesis is introduced, more attention is paid to regulatory mechanism of growth of tumor vessel and anti-angiogenesis. One of important approaches to control the growth and aversion of tumor is to inhibit angiogenesis. Due to its complicated constituents, traditional Chinese medicine (TCM) may influence angiogenesis of tumor in multi targets and diverse level. Searching for high efficient and low toxic inhibitors of angiogenesis, especially from TCM, has become an important issue in worldwide. In present paper, Hirudo and Mylabris were choose to research for their inhibitory effect of angiogenesis. ObjectiveTo investigate the effects of Hirudo and Mylabris on angiogenesis of Chick embryo chorioallantoic membrane (CAM), on human umbilical rein endothel ial cell (HUVEC), and on angiogenesis of transplanted sarcoma S180 in mice. The undergoing mechanisms of inhibitory angiogenesis of Hirudo and Mylabris were studied through detections of vascular endothelial growth factor (VEGF), matrix metalloproteinase (MMP), and micro-vascular density (MVD). It is expect to provide evidences for prevention of invasion and diversion of tumor of Hirudo and Mylabris through this research.MethodsCAM technique and immunohistochemistry were used to research effects of drug-containing serum of Hirudo and Mylabris on CAM angiogenesis, survival of HUVEC in vitro and transplanted sarcoma S180 in mice.1. Experiments in vivo:To observed effects of drug-containing serum of Hirudo and Mylabris on number and area of blood capillary of CAM, and effects on tumor weight and inhibition rate of tumor on S180 ascites tumor mice models. Expression of MMP and VEGF, MVD value were detected with immunohistochemistry.2. Experiments in vitro:Morphological analysis and MTT assay were used to study effects of drug-containing serum of Hirudo and Mylabris on proliferation of cultured HUVEC.Results1. Hirudo and Mylabris had inhibitory effect on number and area of new vessel of CAM blood vessel models of 6 days. Compared with control group, new vessel number of Hirudo high dosage group, Mylabris high dosage and low dosage group was statistically decreased (P<0.01), and vessel area of Mylabris high dosage group was statistically decreased (P= 0.0015). There was no significant difference in number and area of new vessel between control group and serum group (P= 0.9003).2. Compared with control group, thymus gland weight and index in Hirudo high dosage and low dosage group, Mylabris low dosage group were statistically increased (P<0.01); MVD of Hirudo high dosage group and Mylabris high dosage and low dosage group were statistically decreased (P= 0.0252,0.0024,0.0483); VEGF expression in Hirudo high dosage group and Mylabris high dosage and low dosage group were statistically decreased (P= 0.0145,0.0420,0.0023). MMP-9 expression in treatment groups was statistically lower than that in normal control group (P= 0.0011,0.0016,0.0224,0.0078)3. Compared with normal control group, OD value of Mylabris high dosage, medium dosage, low dosage group were statistically decreased(P= 5.7706E-09,0.0003534,2.3148E-06), OD value of Hirudo high dosage and medium dosage group were statistically decreased (P= 1.2854E-Q5,0.0250).Cell morphology observation result: normal cell shape was showed in normal control group,Cells were integrity, and presented spindle shape, and apoptosis was not found. Cell presented shrinkage, even rupture or death, and density of cell was obviously decreased in Hirudo high dosage and medium dosage group, Mylabris high dosage, medium dosage and low dosage group.Conclusion1. Hirudo and Mylabris have inhibitive effect on blood capillary of CAM to a certain extent.2. Hirudo and Mylabris can inhibit the growth of S180 ascites tumor mice, decrease expressions of VEGF and MMP-9 and MVD value in tissue of S180 ascites tumor.3. Hirudo and Mylabris can effectively inhibit proliferation of HUVEC.Hirudo and Mylabris have inhibitive effect on angiogenesis of tumor through decreasing expressions of VEGF, MMP-9, value of MVD and proliferation of HUVEC, which provide theoretical and experimental foundation for inhibiting angiogenesis and anticancer effect of TCM.更多还原