【作者】 张玲；

【导师】 杨向红；

【作者基本信息】 中国医科大学 ， 病理学与病理生理学， 2008， 博士

【摘要】 前言新生血管形成对于恶性肿瘤的无限制生长和转移有重要意义。恶性实体瘤主要由肿瘤细胞和肿瘤间质两部分组成,而肿瘤的发生、发展和转移与间质中血管的生长密切相关。当肿瘤生长到直径1-2mm（约含10⁶个细胞）时,如果没有血管形成,肿瘤细胞将保持静止状态或发生退化、停止增殖甚至死亡。据此理论Folkman提出“抑制肿瘤血管生成”可作为治疗实体瘤的新途径假说。近20年来,抑制肿瘤血管生成已成为控制肿瘤生长及浸润转移研究的一个重要方面。血管抑素（Angiostatin）是近年来发现的第一个内源性肿瘤血管生成抑制因子,因能直接特异地作用于血管内皮细胞,抑制其增殖、减少肿瘤血管生成而倍受青睐。尽管国内外对血管抑素的研究报道很多,但迄今血管抑素的作用机制尚未完全解明,血管抑素作用于血管内皮细胞的信号途径尚未阐明。已有的研究结果提示,血管抑素抗肿瘤血管形成的机制是多方面的,血管抑素可通过抑制内皮细胞增殖、迁移、诱导其凋亡等途径来达到抗血管生成效应,进而起到抑制肿瘤的生长、浸润和转移的作用。为了确切评价血管抑素的抗新生血管形成作用,阐明血管抑素抗新生血管形成的作用机制,我们在人体材料、动物实验及体外细胞培养三个层面上探讨肿瘤组织中血管抑素的表达与肿瘤血管形成的关系及与血管生成调节因子VEGF之间的联系,明确血管抑素对肿瘤生长的影响,进而探讨血管抑素对内皮细胞增殖、凋亡调节的信号传导途径及其作用的分子机制,为肿瘤的防治提供实验依据。实验材料与方法一、人体材料:采用RT-PCR方法通过检测人肺癌组织中Angiostatin与VEGFmRNA的表达及其与微血管密度的关系,明确其在肺癌的发生、发展过程中的作用及其与临床病理之间的关系。二、动物实验:采用人肺癌A₅₄₉细胞建立c57BL/6近交系小鼠移植瘤模型进行实验,评价Angiostatin治疗肺癌的有效性。48只荷人肺癌A₅₄₉ C57BL/6近交系小鼠（皮下移植瘤）分成4组,分别皮下肿瘤内注射Angiostatin（浓度分别为0.6mg/Kg、1.2mg/Kg、2.4mg/kg）,对照组注射等量生理盐水。注射次数1次/2d,治疗14天,观察肿瘤重量的变化;并用常规病理方法和免疫组化S-P法检测肿瘤组织中细胞的形态学改变和CD34、Survivin和NF-κB的表达,计数微血管密度（MVD）,观察Angiostatin对人肺癌小鼠移植瘤新生血管形成的影响。三、体外培养:在体外研究中培养HUVECs和人肺癌A₅₄₉细胞,应用MTT比色法检测血管抑素对HUVECs和A₅₄₉细胞增殖活性的影响,台盼兰染色细胞计数法观察血管抑素对人肺癌A₅₄₉和血管内皮细胞生长的影响;建立肿瘤微血管模型观察血管抑素对内皮细胞管腔形成的影响;流式细胞术分析血管抑素对HUVECs细胞生长周期的影响。通过蛋白印记法检测血管抑素对人血管内皮细胞作用的信号传导途径及其相关因子的表达,初步探讨血管抑素抗新生血管形成作用的分子机制。结果一、人体材料检测结果:肺癌组织中Angiostatin mRNA表达量为5.56±1.40,癌旁肺组织mRNA表达量为7.85±1.74;VEGF mRNA表达量癌组织为12.43±1.58,癌旁组织为9.10±1.20。组间比较差异显著（P＜0.05）;Angiostatin mRNA在Ⅰ-Ⅱ期肺癌组织中表达量为9.41±2.29,在Ⅲ-Ⅳ期肺癌组织中表达量为5.26±2.39,后者表达水平较前者明显降低,差异显著（P＜0.05）;有淋巴结转移组的Angiostatin mRNA表达水平低于无转移组,差异显著（P＜0.05）。VEGF mRNA在Ⅰ-Ⅱ期肺癌组织中表达为9.79±1.45,在Ⅲ-Ⅳ期肺癌组织中表达为16.83±2.38,后者表达水平明显高于前者,差异极显著（P＜0.01）;在低分化组表达与高-中分化组相比有显著性差异（P＜0.05）;淋巴结转移组表达高于无转移组,差异极其显著（P＜0.01）。Angiostatin与VEGF mRNA在NSCLC中表达的相关性检验结果r=-0.671,P＜0.05,二者呈负相关。二、动物实验显示:（一）肿瘤重量和抑瘤率的检测:随着接种瘤细胞时间的增加,各组小鼠肿瘤都逐渐增大,但Angiostatin治疗组肿瘤生长明显缓慢。与对照组比较,Angiostatin三个剂量组均有抑瘤作用,其抑制程度最高达77%（p＜0.05）;（二）肿瘤微血管密度的检测:对照组移植瘤组织CD34弥漫分布;统计学分析显示Angiostatin治疗组肿瘤的微血管密度下降,随着Angiostatin剂量增加MVD数量减低,治疗组与对照组相比有显著性差异（p＜0.05）。（三）survivin和NF-κBp65的表达:阳性反应产物分布于瘤细胞或内皮细胞的胞浆与胞核内,呈棕黄色。Survivin蛋白在各组的阳性率分别为:对照组100%（12/12）,Angiostatin小剂量组91.6%（11/12）,中剂量组75%（9/12）,大剂量组66.6%（8/12）;NFκBp65在对照组和Angiostatin小剂量组表达的阳性率相同,为33.3%（4/12）,中剂量组16.7%（2/12）,大剂量组25%（3/12）;图象分析结果显示,血管内皮细胞NFκBp65表达对照组为0.313±0.025,对照组Survivin表达则为0.295±0.027,二者随Angiostatin剂量加大其表达逐渐降低;且有统计学意义（P＜0.05）。三、体外细胞实验:（一）MTT检测结果显示:不同浓度的血管抑素作用于人脐静脉内皮细胞24h后,OD₄₉₀吸光度值均低于对照组,对内皮细胞增殖的抑制作用随着血管抑素浓度的增加而增强,与对照组相比差异均有显著性意义（P＜0.01）。并且随着血管抑素作用时间的延长,细胞增殖明显受抑制,吸光度值均低于对照组。72h时最大抑制率达到91.37%,且与对照组相比差异均有显著性意义（P＜0.01）。（二）流式细胞术检测细胞周期:不同浓度Angiostatin组与对照组比较,G₁期细胞比例有所减少,S期有所增加。从4mg/L血管抑素开始出现凋亡,到16mg/L血管抑素时出现了明显的凋亡峰,而且随着剂量增大,凋亡渐强。（三）内皮细胞管腔形成实验:人脐静脉内皮细胞接种于鼠尾胶后,以肿瘤上清液诱导,内皮细胞从散在分布状态逐渐聚集、形成条索状,12h形成明显的网状。在培养人脐静脉内皮细胞3h后（血管网未形成之前）加入血管抑素,未见内皮细胞形成网状结构;内皮细胞培养24h后（血管网已形成）加入血管抑素,8h内皮细胞成网数量减少,变圆,逐渐聚集,12h已形成的血管网状结构破裂,并在断裂后消失。（四）流式细胞术检测内皮细胞整合素αvβ3的表达:对照组平均荧光表达是6.76±2.19,单纯bFGF组42.3±4.41,Angiostatin低剂量组是24.8±4.26,高剂量组为2.34±1.16。两两比较则有明显的统计学意义。表明Angiostatin抑制了bFGF诱导的内皮细胞整合素αvβ3的表达。（五）Western Blot法检测Angiostatin对内皮细胞血管生成相关信号通路的影响:以特异性抑制剂作用后,Akt、P38、FAK的蛋白表达水平较低,bFGF刺激后略有增高。经Angiostatin处理后的内皮细胞中NF-κBp65、Survivin表达水平随Angiostatin剂量增加表达减低。结论1、Angiostatin在人非小细胞性肺癌中的表达与肿瘤的临床分期、分化程度及淋巴结转移有关;NSCLC中Angiostatin与VEGF的表达呈明显的负相关。2、Angiostatin对人A₅₄₉肺癌小鼠移植瘤生长及新生血管形成具有抑制作用。3、Angiostatin能抑制体外培养人血管内皮细胞的增殖、诱导其凋亡,并抑制肿瘤诱导的体外管腔形成。4、Angiostatin对内皮细胞的抑制增殖或诱导凋亡的作用与整合素avβ3的表达及Survivin、NF-κB有关。更多还原

【Abstract】 Introduction and ObjectiveNeovascularization for unlimited malignant tumor growth and metastasis has important significance.Malignant solid tumors mainly composed of two parts,that is the tumor cells and stromal tumor.And the occurrence,development and transfer of tumor closely related to the growth of blood vessels in supportive stroma.When the tumor grew to 1-2 mm in diameter（containing about 106 cells）,if there is no nutrition and oxygen supply vessels,the tumor cells will remain dormant or degradation occurred, and stop the proliferation or even death.With this theory,Folkman puts forward the hypothesis of "inhibit tumor angiogenesis" as a new ways of treatment for solid tumors. Over the past 20 years,the inhibition of tumor angiogenesis has become an important aspect of research in the control tumor growth,invasion and metastasis.Angiostatin is the first in recent years found that endogenous tumor angiogenesis inhibitor,which specifically affect directly vascular endothelial cells,blocking the proliferation and migration,sequentially reducing rumor angiogenesis.Although there are a lot of research reports about angiostatin at home and abroad,the mechanism of antiangiogenic action of angiostatin has not explained fully so far,the cell signaling pathway of angiostatin acting on the vascular endothelial cells has not been proven. Preliminary results of the study have revealed the mechanism angiostatin anti-tumor angiogenesis is a multi-factor.Angiostatin can inhibit proliferation,migration of endothelial cells,and induce apoptosis of endothelial cells to achieve anti-angiogenesis effect,thereby inhibiting tumor growth,invasion and metastasis.In order to accurately evaluate the action of anti-neovascularization and understand of anti-angiogenesis mechanism about angiostatin,we investigate the relationship between expression of angiostatin and tumor angiogenesis in the tumor tissue and the regulation of angiogenesis factor VEGF link in the body material,animal and in vitro cell culture experiments at three levels.And further definitude the impact of angiostatin on tumor growth and the major signaling pathway on angiostatin inhibiting proliferation of endothelial cell and apoptosis regulation,simultaneity explore the molecular mechanism of its role.These researches may provide some theoretical references and foundational understandings for the prevention and treatment of tumors.Materials and Methods1.Human materialsRT-PCR was used by detecting expression of VEGFmRNA and angiostatin mRNA in human lung carcinoma and relationship between its and Microvascular density,elucidating role of angiostatin in the process of occurrence and development of lung carcinoma and relationship between its and clinical pathology.2.Animal experimentsA₅₄₉ human lung carcinoma cells were used by establishing c57BL / 6 inbred mouse xenograft model experiments to evaluate the effectiveness of Angiostatin treatment of lung carcinoma.48 of human lung carcinoma A₅₄₉ C57BL / 6 inbred mice （subcutaneous xenografts）be divided into four groups,12 mice each groups,three groups are experiment,one is control.Subcutaneous injection Angiostatin0.2ml （concentration of 0.6 mg/ Kg,1.2 mg/ Kg,2.4 mg / kg） in the tumor of mice.Control group was injected with normal physiological brine equate.Injection number 1 / 2 d, and 14 days of treatment,tumor changes was observed.And using conventional pathological and immunohistochemical S-P methods were used by detecting of cellular morphological changes and CD34,Survivin and NF-κB expression in the tumor tissue, counting microvascular density（MVD）,observating Angiostatin’s effect on human lung carcinoma in mice transplanted tumor angiogenesis.3.Cells culture in VitroUsing primary cells cultured mathods culture human umbilical vein endothelial cells and A₅₄₉ human lung carcinoma cells in the study in vitro.Angiostatin impacting on proliferation activity of HUVECs and A₅₄₉ human lung carcinoma cells are examined by MTT colorimetric assay.Trypan blue stain cells counting was used observed angiostatin’s influence on the growth of A₅₄₉ human lung carcinoma and vascular endothelial cells.Establishing of tumor microvascular model was used to observed angiostatin’s role for endothelial cells shaping vascular cavity.With the flow cytometry,angiostatin on the impact on HUVECs cell cycle was analysed.Through western blot,we detect angiostatin imprinting of understandings on the role of vascular endothelial cell signal transduction pathway and its correlative factors,preliminary study the molecular mechanism of angiostatin anti-angiogenesis.Results1.Detection of human materials:Angiostatin mRNA in lung carcinoma tissue for the expression of 5.56±1.40, adjacent lung tissue for the expression of mRNA 7.85±1.74;VEGF mRNA expression in lung carcinoma tissues for 12.43±1.58,adjacent lung tissues to 9.10±1.20. Difference between groups was significant（P＜0.05）;Angiostatin mRNA inⅠ-ⅡPhase lung carcinoma tissue for the expression of 9.41±2.29,Ⅲ-Ⅳlung carcinoma tissue for the expression of 5.26±2.39,the latter than the former obvious expression level lower,the difference was significant（P＜0.05）;metastasis group angiostatin-mRNA expression levels lower than those without metastasis group,the difference was significant（P＜0.05）.VEGF mRNA expression inⅠ-ⅡPhase lung carcinoma 9.79±1.45,expression inⅢ-Ⅳlung carcinoma for 16.83±2.38,the latter expression levels were significantly higher than the former,the difference was extremely significant（P＜0.01）;In cancer Section of the poorly differentiated cells and high - compared differentiation,there was a significant difference（P＜0.05）,expression in the lymph node metastasis group was higher than that without lymph node metastasis group,the difference is extremely significant（P＜0.01）.2.Animal experiments showed that:（1） Weights and inhibitory rate of tumorTumor cells inoculated with the increase in time,the tumors each groups of mice are gradually increasing,but the angiostatin treatment groups significantly slow tumor growth.Compared with the control group,three dose groups of angiostatin have anti-tumor effect averagly,the maximum inhibition of 77%（p＜0.05）;（2） Microvessel dentisy of tumorCD34 expression:there all have expression in control group and tumor treatment groups.Under the microscope,CD34 diffuse distribution,in brown,yellow positive cells;Statistical analysis showed that tumor microvessel density of angiostatin treatment groups decreased,with dose angiostatin increasing MVD reduce.Compared with the treatment groups and control group,there were significant differences（p＜0.05）.（3） Expression of survivin and NF-κBThe positive reaction products of survivin and NF-kB distribute in the nucleus and cytoplasm of the tumor cells,was brown.The positive rates in each group of survivin protein were as follows:control group,100%（12/12）,angiostatin small dose group 91.6%（11/12）,in the middle dose group 75%（9/12）,66.6%of high-dose group（8/12）; The positive rates of NF-kBp65 in the control group and angiostatin low dose group,is the same expression,for 33.3%（4/12）,16.7%in the dose group（2/12）,high-dose group was 25%（3/12）;Image analysis showed the expression of NF-kBp65 in control group of vascular endothelial cell is 0.313±0.025,the expression of survivin in control group of vascular endothelial cell is 0.295±0.027.With Angiostatin dose increase,the expression of NF-kBp65 and Survivin in endothelial cell decreased gradually. Compared with the control group there is statistical significance（P＜0.05）.3.experiments in vitro:（1） MTT assay results showed that:Different concentrations of angiostatin act in human umbilical vein endothelial cells after 24 h,OD₄₉₀ absorbance values were lower than the control group,its inhibiting action on the proliferation of endothelial cells with increasing concentrations of angiostatin increased,compared with the control group differences were significant （P＜0.01）.And along with extension of acting time,the proliferation of human umbilical vein endothelial cells inhibited significantly,its absorbance values were lower than the control group.Measurement results show that:when 72h,the maximum inhibition rate reached 91.37 percent,compared with the control group the difference was significant（P＜0.01）.（2） Flow test results of the cell cycle:Compared with different concentrations Angiostatin group and the control group, the proportion of cells in G1 phase has declined,in S phase has increased.From 4 mg/L angiostatin on,apoptosis began,and to 16 mg/L angiostatin,there is a clear peak of apoptosis,and increased with the dose,apoptosis peak is gradually strong.（3） Test of cavity formation in endothelial cells:After human umbilical vein endothelial cells were cultured in Tail plastic of mice and induced by tumor supernatant,endothelial cells of morphology have been observed from scattered in the state to gathered,gradually cord-like dendritic formation,12h reticular formation obvious.In cultured human umbilical vein endothelial cells after 3 h （vascular network has not formed before） by adding angiostatin,endothelial cells have not formed network structure.When endothelial cells were cultured for 24h（vascular network has been formed） by adding angiostatin,we discovered 8h endothelial cells decrease in the number of formating networks and became round,and gradually gathered,12h blood vessels network structure formed by endothelial cells have been ruptured,and disappear after fracture,the remnants of the endothelial cells form cells lumped together.（4） Flow detection expression of integrinαvβ3 in endothelial cells:FCM results showed,the average fluorescence density expression in control group is 6.76±2.19,pure bFGF group is 42.3±4.41,Angiostatin low dose group is 24.8±4.26,the high-dose group of angiostatin is 2.34±1.16.Comparing groups,there is a relatively obvious statistical significance.This results indicate that angiostatin restrained the expression of integrinαvβ3 in endothelial cells by bFGF induction.（5） Western Blot detection Angiostatin role after the angiogenesis pathwayAfter reaction of endothelial cells and spicific signal pathway inhibitors Akt,P38, FAK protein expression levels are relatively low,bFGF stimulated slightly higher.At the same time in the endothelial cells after treated with Angiostatin,expression levels of NF-κBp65 and Survivin increased with the dose of Angiostatin reduced.Conclusions1.The expression of Angiostatin in human non-small cell lung carcinoma correlate with the clinical tumor stage,differentiation and lymph node metastasis.2.Angiostatin can restrain the growth on A₅₄₉ lung carcinoma in mice transplanted tumor and inhibit neovascularization.3.Angiostatin can inhibit the proliferation of human vascular endothelial cell incubated in vitro and induce its apoptosis,and restrain blood vessel cavity formation induced by tumor in vitro.4.Angiostatin related with inhibition of endothelial cell proliferation or induction of apoptosis and the role of the integrin avβ3 expression and Survivin,NF-κB.更多还原