【作者】 李伟；

【导师】 潘运龙；

【作者基本信息】 暨南大学 ， 胃肠外科， 2017， 博士

【摘要】 实体肿瘤的生长和肿瘤血管生成(Angiogenesis)密切相关,肿瘤细胞的快速增殖所导致的相对缺氧,使肿瘤组织持续产生促血管生成因子,破坏了原有的促-/抗血管生成平衡的状态,导致肿瘤血管具有明显的异质性,使肿瘤微环境恶化,增加了恶性程度并降低治疗效果。肿瘤血管已成为肿瘤治疗的重要靶点,通过短暂的抗血管治疗使异常的肿瘤血管正常化,联合抗肿瘤治疗,获得了显著的效果。目的:首先观察纳米金(Gold nanoparticles,AuNPs)联合抗血管药物重组人血管内皮抑素(Recombinant human endostatin,rhES)的肿瘤靶向性,并检测其所诱导的肿瘤血管正常化的时间窗,以及是否可以提高常规化疗的效果;其次观察纳米金对肿瘤血管正常化和肿瘤转移的影响,并探讨其相关机制;最后观察血小板反应蛋白-1(Thrombospondin-1,TSP-1)在肿瘤血管正常化过程中的变化趋势,以确定其是否可以作为一种血管正常化的监测指标。方法:第一章,通过紫外分光光度法、动态激光散射和场发射透射电子显微镜鉴定纳米金和重组人血管内皮抑素结合前后的特性,通过免疫荧光技术检测药物的靶向性,血管正常化过程中周细胞覆盖率(α-SMA/CD31)、血流灌注(FITC-Lectin/CD31)、血管通透性(FITC-Dextran/CD31)和肿瘤组织缺氧诱导因子(hypoxia inducible factor-1α,HIF-1α)的表达;在正常化区间内给予5-FU(40 mg/kg),通过高效液相色谱(High Performance Liquid Chromatography,HPLC)检测肿瘤组织中5-FU的浓度,并观测肿瘤大小和荷瘤模型的中位生存时间(Median survival time,MST)。第二章,通过C57BL/6后肢足垫注射B16F10细胞建立黑色素瘤荷瘤模型,给予纳米金(1mg/kg)处理,通过免疫荧光检测血管通透性和灌注率,利用免疫组织化学检测肿瘤上皮-间质转化(epithelial-mesenchymal transition,EMT)、组织缺氧程度(哌莫硝唑染色法)和组织中血管内皮生长因子(vascular endothelial growth factor,VEGF)及其受体(VEGFR2);通过细胞划痕实验检测纳米金对肿瘤细胞和内皮细胞迁移的影响,通过CCK-8法检测对细胞活性的影响,利用蛋白质印迹检测基质金属蛋白酶-2(MMP2),c-Myc,波形蛋白(Vimentin),E-钙黏蛋白(E-Cadherin),紧密连接蛋白(ZO-1)和富含半胱氨酸的酸性分泌蛋白(SPARC)在肿瘤细胞和脐静脉内皮细胞中的变化,利用实时荧光定量核酸扩增(Real-time Quantitative PCR,RT-qPCR)检测EMT相关基因表达变化。第三章,通过皮下注射SW620细胞至Balb/c裸鼠皮下建立结直肠癌荷瘤模型,免疫荧光和免疫组化确定肿瘤血管正常化的出现,结合酶联免疫吸附实验(ELISA)检测外周血中人血小板反应蛋白-1(Thrombospondin-1,TSP-1)随时间窗变化的趋势。结果:首先纳米金与重组人血管内皮抑素结合后明显提高了后者在肿瘤内的剂量,并且维持了较长的时间。免疫荧光显示用药后的第4天和第8天肿瘤血管呈现正常化的表现:血管周围α-SMA覆盖增加,FITC-Lectin灌注增加,FITC-Dextran在血管外周沉积减少,肿瘤组织HIF-1α表达降低,在正常化时间窗内给予5-FU治疗,提高了5-FU在肿瘤内的浓度,明显减小了肿瘤体积,延长了荷瘤模型的中位生存时间。其次,单纯使用纳米金增加了FITC-Lectin在肿瘤组织中的灌注,减少了FITC-Dextran的渗出,哌莫硝唑染色显示处理组的缺氧程度缓解;免疫组化显示组织中Vimentin的表达降低,而E-Cadherin的表达升高,证明对EMT过程具有抑制作用。纳米金降低了肿瘤组织中VEGF的表达,但VEGFR2未见明显抑制。细胞实验表明纳米金抑制了肿瘤细胞和内皮细胞的迁移,但CCK-8结果显示两种细胞的活性并未受影响;蛋白质印迹检测纳米金抑制内皮细胞MMP2表达,促进ZO-1的表达,抑制了肿瘤细胞MMP2、c-Myc和SPARC的表达,逆转肿瘤细胞中Vimentin和E-Cadherin的比值。RT-qPCR结果显示与EMT相关的Snail-1,Snail-2,Twist-1,ZEB-1和ZEB-2表达下降。最后,通过ELISA我们发现外周血中TSP-1在肿瘤血管正常化期间明显的降低,并且该趋势与肿瘤血管正常化的时间窗相符合。结论:1.纳米金可作为药物载体靶向输送重组人血管内皮抑素进入肿瘤组织;2.纳米金结合重组人血管内皮抑素短暂治疗促进了肿瘤血管正常化,增加了化疗效果,并且具有明显的时间相关性;3.纳米金通过下调SPARC、MMP-2和c-Myc以及相关基因的表达抑制肿瘤细胞EMT的发生,并降低了黑色素瘤的肺转移。4.纳米金通过下调MMP-2抑制内皮细胞迁移和血管形成能力,并通过上调ZO-1的表达增强血管壁的完整性;5.血浆中TSP-1的变化与肿瘤血管正常化时间窗内缺氧程度的变化趋势相符,有希望成为预测肿瘤血管正常化的一个指标。更多还原

【Abstract】 Angiogenesis is a process by which vessels are formed through preexisting ones,and this plays a key role in the progression of solid tumors.This process is initiated by the wealth of pro-angiogenic factors produced by tumor cells in response to a hypoxic microenvironment.Excessive production of pro-angiogenic factors leads to an imbalance between pro-and anti-angiogenic signals,which results in structurally and functionally abnormal vessels characterized by dilatation,tortuosity,high permeability,risk of recurrence or metastasis,and reduction of radiotherapy or chemotherapy effect.Moderate anti-angiogenic therapies are considered to regulate tumor vascular morphology and function and improve the efficiency of antitumor therapy.However,the duration of vascular normalization is transient.Thus,monitor the process from start to finish is crucial and the identification of markers that indicate normalization may be useful to determine the optimal schedules on which to administer anti-tumor therapies.Objective: First,to investigate the targeting of gold nanoparticles(AuNPs)combined with anti-angiogenesis agents,and detect the time window of tumor vascular normalization and anti-tumor effect of 5-FU.Second,observe the effect of AuNPs on tumor vascular normalization and the inhibition of metastasis,and to explore its related mechanisms.Finally,observe the changes of Thrombospondin-1 during the time window of tumor vascularization.Methods: In the first section,the characterization of AuNPs with recombinant human endostatin(rhES)was identified by ultraviolet-visible spectrophotometry(UV-vis spectrum),dynamic laser scattering(DLS)and field emission transmission electron microscopy(FETEM).The dose of rhES in tumors,pericyte coverage(α-SMA / CD31),blood perfusion(FITC-Lectin / CD31),vascular permeability(FITC-Dextran / CD31)and the expression of hypoxia inducible factor-1α(HIF-1α)in tumor tissue were measured by immunofluorescence technique.5-FU was given in the normalization window and the concentration of 5-FU in tumor tissue was detected by high performance liquid chromatography(HPLC).The tumor size and median survival time(MST)of tumor bearing model was observed.In the second section,tumor vascular permeability(FITC-Dextran)and perfusion(FITC-Lectin)were measured by immunofluorescence,the epithelial-mesenchymal transition(EMT),vascular endothelial growth factor(VEGF)and its receptor(VEGFR2)and alleviation of tumor hypoxia(pimetanazole staining)were detected by immunohistochemistry.The effect of AuNPs on migration of tumor cells and endothelial cells was detected by wound healing assay,and the the cell viability were detected by CCK-8 method.The expression of MMP2,c-Myc,Vimentin,E-Cadherin,ZO-1 and SPARC in tumor vascular normalization and metastasis were observed by Western blotting.In the third section,the normalization of tumor blood vessels was identified as as described above,the change of thrombospondin-1in peripheral blood during time window was measured by enzyme-linked immunosorbent assay(ELISA).Results: The accumulation of rhES in tumor significantly increased after combined with AuNPs,and maintained a long time compared with the control.Immunofluorescence showed normalization of tumor blood vessels from day 4 to day 8 after AuNPs-rhES administration,the coverage of pericyte(α-SMA)and FITC-Lectin perfusion were increased,the deposition of FITC-Dextran around the blood vessels and the expression of HIF-1α in tumor tissue were decreased.Compared with monotherapy,combination with AuNPs-rhES significantly increased 5-FU localization to the tumor sections.5-FU monotherapy and 5-FU combined with AuNPs-rhES reduced growth significantly,however,tumors receiving combination therapy exhibited a more significant reduction in growth compared to tumours receiving 5-FU monotherapy.Our results from the lectin analysis revealed that tumor blood perfusion significantly increased after Au NPs treatment compared with controls,and the ratio of lectin+ to CD31 expression indicated vessels with regular perfusion.Leakage of injected dextran into tumor vasculature remarkably decreased after AuNPs treatment.The immunohistochemical staining showed that the AuNPs treatment significantly reduced tumor hypoxia,and down-regulated the expression of Vimentin and up-regulated the expression of E-Cadherin in the tissue.AuNPs reduce the extent of lung metastasis in vivo,and suppress the migration of HUVECs and B16F10 cells in vitro.On the contrary,data from the CCK-8 test indicated that the AuNPs had no effect on the cell viability.In addition,AuNPs inhibits the expression of MMP2 in endothelial cells,but promotes the expression of ZO-1,inhibits the expression of MMP2 and c-Myc in tumor cells,reverses the process of EMT.RT-qPCR showed that AuNPs reduced the expression of Snail-1,Snail-2,Twist-1,ZEB-1 and ZEB-2.Finally,we found that the expression of plasma thrombospondin-1 decreased significantly during normalization of tumor vascular normalization(day 2-8),and then increased after the closure of normalization window.Conclusion:1.AuNPs can be used as a nano-drug delivery system to target transport rhES into tumor tissue.2.The combination of AuNPs with rhES promoted the normalization of tumor blood vessels,and increased the effect of chemotherapy.3.AuNPs inhibit EMT in tumor cells by down-regulating MMP-2,c-Myc,SPARC and EMT related genes.4.AuNPs inhibit endothelial cell migration and angiogenesis by down-regulating MMP-2 and enhance vascular wall integrity by up regulating the expression of ZO-1.5.The change of circulating thrombospondin-1 is consistent with the hypoxia in the time window of tumor vascular normalization,and it is expected to be an indicator to predict the normalization of tumor blood vessels.更多还原