【作者】 王桂华；

【导师】 唐劲天； 廖遇平；

【作者基本信息】 中南大学 ， 肿瘤学， 2008， 博士

【摘要】 研究背景:恶性肿瘤的发病率呈现逐年升高的趋势,在许多国家已位居常见死亡原因的首位。虽然世界各国都在尽力防治恶性肿瘤这类疾病,但是进步不快。肿瘤治疗问题已经有了很多苗头和好的趋向,治愈率也有一定提高,但仍然需要从整体策略方面加以改善。人们迫切期望治疗手段有重大的突破,以达到大幅度降低发病率和提高治愈率的目的。传统的肿瘤加温治疗已有多年历史,是继手术、放疗、化疗和生物治疗之后的又一个重要的肿瘤治疗措施,但一直处于辅助和姑息治疗的地位,其主要问题是肿瘤治疗温度太低。近年来,纳米技术的研究获得了飞速发展,磁性纳米微粒不仅可以用作药物载体,而且在一定强度和频率的交变磁场作用下可发热升温。将磁性纳米材料（magnetic nanoparticle,MN）导入肿瘤病灶局部,可以精确地将靶组织加热到更高的治疗温度,实现所谓的肿瘤“适形热疗”（comformalhyperthemia,CH）,已成为研发肿瘤热疗新方法的重要研究方向和可能的突破口。热刺激导致的组织热效应包括直接热效应和间接热效应。前者被认为主要是细胞的坏死和凋亡,而后者主要是机体的免疫反应和类似通过细胞因子影响血管生成等,可能在肿瘤的热治疗中发挥更重要的作用。间接热效应分子生物学机制可能包括:巨噬细胞活化、多种细胞因子释放、梗塞再灌注损害和免疫反应等。根据我们的文献检索分析,尚未见关于间接热效应的血管生成机制方面的研究报道。为此,我们采用了基于磁性纳米技术的磁感应治疗新方法,对影响肿瘤血管生成的生物学效应进行了初步探讨。第一章Wistar大鼠Walker-256肿瘤模型的建立及生物学特性目的:探讨建立Wistar大鼠皮下Walker-256肿瘤模型的可行性,并观察其生物学行为。方法:Wistar大鼠右腋皮下注射Walker-256肿瘤细胞悬液2×10⁶个细胞/0.6ml·只,建立皮下肿瘤模型,观察肿瘤生长情况,并取皮下肿瘤结节进行病理检查。结果:1)7～9天左右种植部位皮下即可长出直径约1.0cm的肿瘤结节,病理证实为恶性肿瘤。2)肿瘤种植成功率高,达97.78%（176/180例）。3)荷瘤大鼠平均自然生存期较长,达35.50±5.59天。4)动物肿瘤模型稳定,肿瘤结节直径达到0.5～1.0cm后,肿瘤无自发消退现象。结论:1)建立Wistar大鼠皮下Walker-256肿瘤模型成瘤率高,可作为恶性肿瘤基础研究的模型;2)所建立的模型生物学性状稳定,荷瘤生存期较长,肿瘤无自发消退现象。第二章Fe₃O₄纳米磁流体介导磁感应治疗Wistar大鼠Walker-256皮下肿瘤目的:探讨Fe₃O₄磁性纳米微球磁感应加热治疗Wistar大鼠Walker-256皮下肿瘤的作用。方法:用Walker-256肿瘤细胞株建立Wistar大鼠皮下移植瘤模型,将研制的Fe₃O₄磁性纳米微球注入肿瘤组织内,加入交变磁场中进行磁感应治疗,控温在50～55℃,分别加热治疗1、2、3次（即MFH1、MFH2和MFH3组）后,分析比较3个磁感应治疗实验组和注入磁流体未热疗组（MF组）较生理盐水对照组（NS组）的肿瘤体积抑制率和生存期。同时观察磁感应治疗后肿瘤的病理改变,以及磁流体介导磁感应治疗的升温效果。结果:1)肿瘤组织温度能在6～10分钟内达到50～55℃,周围正常组织不升温。2)磁流体介导的磁感应治疗对MFH2和MFH3组肿瘤在2周内有明显的肿瘤体积抑制作用,肿瘤体积抑制率65±3.5%、71.6±4.2%。治疗对MFH1组肿瘤也有一定抑制作用,但曲线斜率改变不明显。MF组肿瘤体积一时间曲线无差别。3)NS组平均生存期35.50±5.59天,MFH3组:41.88±6.48天,MFH2组:42.13±4.98天,MFH1组:39.00±5.30天,MF组:36.06±6.57天。与NS组比较:MFH3和MFH2的生存期延长,差异有统计学意义,q值分别为-6.375,-6.625;P值分别为0.003,0.002。MFH1和MF的生存期差异无统计学意义,q值分别为-3.500,-0.563;P值分别为0.093,0.785。4)NS组、MF组肿瘤表面光滑,或见少许出血,小灶性坏死区。癌细胞密度大,细胞核大,核仁明显,易见核分裂相。热疗组部分肿瘤表面凹凸不平及焦痂。肿瘤组织内大片坏死残留的无结构红染物质,或空洞,部分细胞核有固缩、碎裂及崩解,可伴有明显出血区（MFH3、MFH2比MFH1组明显）。结论:1)Fe₃O₄的磁流体热疗能使靶组织达到理想的治疗温度,而正常组织不升温,即“适形热疗”。2)磁流体热疗能显著抑制Wistar大鼠Walker-256皮下肿瘤增殖,延长荷瘤鼠的生存期,促进肿瘤细胞凋亡。3)对于肿瘤的抑制效应,存在明显的时间与热剂量关系。4)磁流体热疗对该肿瘤的治疗作用提示,它也可能为其他实体瘤的治疗提供一条新的途径。第三章Walker-256皮下肿瘤模型磁感应治疗后对血管生成的影响目的:探讨磁流体介导的磁感应治疗对Wistar大鼠Walker-256皮下肿瘤模型血管生成的影响。方法:将制作的Fe₃O₄磁性纳米微球注入Wistar大鼠Walker-256皮下肿瘤模型的肿瘤组织内,加入交变磁场中磁感应治疗,温度控制在50～55℃,分别加热治疗1、2、3次（即MFH1、MFH2和MFH3组）后,采用逆转录—聚合酶链反应（RT-PCR）和免疫组化（S-P）法分析比较3个实验组与对照组的血管内皮生长因子（VEGF）及受体（Flt-1、Flk-1）的mRNA水平、VEGF与微血管密度（MVD）的表达水平,并分析VEGF与MVD的相关性。结果:1)MFH3、MFH2组VEGF阳性染色肿瘤细胞累积光密度值（IOD）和MVD阳性染色内皮细胞计数明显减少。与NS组比较,差异有统计学意义,p值分别为0.000,0.001;0.000,0.001。而MF、MFH1组的差异无统计学意义,p值分别为0.999,0.675;0.994,0.332。2)对肿瘤细胞中VEGF表达和间质中内皮细胞MVD计数进行相关性分析,两者有显著相关性,r=0.898,p=0.000。3)磁感应治疗能下调MFH3和MFH2组的VEGF mRNA、Flk-1 mRNA表达,与NS组比较,差异有统计学意义,P值分别为0.000,0.006;0.005,0.014。而MF和MFH1组的VEGF mRNA、Flk-1 mRNA表达无明显改变,差异无统计学意义,P值分别为0.648,0.231;0.994,0.158。4)磁感应治疗能下调MFH3组的Flt-1 mRNA表达,与NS组比较,差异有统计学意义,P=0.009,而MF、MFH1、MFH2组的Flt-1 mRNA表达无明显改变,差异无统计学意义,P=0.954,0.983,0.699。5)在50～55℃范围内,Flk-1受体比Flt-1受体对磁感应热疗的反应更敏感,提示新生血管可能比成熟血管对热剂量更敏感。结论:1)磁流体介导的磁感应治疗能下调荷瘤鼠内血管内皮生长因子（VEGF）及受体（Flk-1和Flt-1）的表达。这些血管生成调控因子的减少,可能导致了肿瘤内血管生成的减少,MVD计数也证实肿瘤内血管数目的下降。2)磁流体热疗对新生血管的抑制作用比成熟血管更大。由此推断:磁流体热疗抑制肿瘤组织内的血管生成,可能主要表现为新生血管减少,即其主要影响新生血管的生成。磁流体热疗的间接热效应可能是其发挥肿瘤治疗作用的更重要途径。更多还原

【Abstract】 Background:Disease incidence of malignant tumor is increasing gradually every year,and malignant tumor becomes the first cause of disease death.Although many works have been done to prevent and cure the malignant tumor in the world wide,improvement is slow.People are eagering for a method to break through this bottle neck so as to cut down disease incidence and elevate cure rate largely.Traditional hyperthermia for malignant tumor has a long history, which is another important tool for curing cancer after surgery, radiotherapy,chemotherapy and biotherapy.However,traditional hyperthermia is an auxiliary and palliative therapy because of its lower therapy temperature.In recent years,studies on nano-hitech have been fastly improved,magnetic nanoparticle can be used as drug vehicle and also produce heat energy under an alternating magnetic field with certain intensity and frequency.Magnetic nanoparticle can be implanted into the tumor tissues and accurately heat the tumor tissues,at the same time,the tissues beside tumor will not be heated,I.e,"comformal hyperthermia", which has been a "heat spot" and potential breach in biomedicine technology to oncology study.Tissue effect caused by focal hyperthermia application occurs in two distinct phases--direct effect and indirect effect.The first phase includes cell death and apoptosis.The second phase is an indirect effect that produces a progressive tissue damage,which may be more important in oncology hyperthermia.This progressive injury may involve a balance of several factors,such as ischemia-reperfusion injury,Kupffer cell activation,altered multi-cytokine expression,and modulation of the immune response,and so on.As we know,there are no reports about angiogeneosis of indirect injury after hyperthermia application. We make use of this magnetic nano-hitech to investigate angiogeneosis mechanism of malignant tumor after focal hyperthermia.If we know much better about the molecular and biological basis of hyperthermia to malignant tumor,we can do much better to prevent and cure malignant tumor.CHAPTER ONE:Establishment and biological characteristics of subcutaneous tumor model of carcinomatous Walker-256 cells in Wistar ratsObjective:To study the feasibility of establishment of subcutaneous tumor model of carcinomatous Walker-256 cells in Wistar rats and its tumor biological characteristics.Methods:Carcinomatous walker-256 cells（about 2×10⁶ cells with 0.6ml per rat） were injected into the right axilla to form subcutaneous tumor model in rats.Then the subcutaneous tumor model’s growth was observed,and tumor node’s pathology examination was made.Results:1) Malignant tumor node whose diameter was 1 centimeter or so would be found after carcinomatous walker-256 cells injected within 7 or 9 days,which was proved by pathology examination. 2) The successful rate of the subcutaneous tumor model was very high, about 97.78%（176/180 cases）.3) The natural survival time of the subcutaneous tumor model in rats was very long,namely 36 days.4) Animal model was stable,and tumor node was never disappear as soon as its diameter was more than 0.5 centimeter.Conclusion:1) The tumor model of carcinomatous walker-256 cells in Wistar rats is an ideal model for studying the mechanism and basis of malignancy,just because of its high successful rate.2) Biologic character of tumor model is steady,and tumor nodes will not spontaneously disappear.The natural survival time of tumor model is enough long to do some experiments.CHAPTER TWO:Study on the therapeutic effect of Fe₃O₄ nanometer magnetic fluid hyperthermia on subcutaneous tumor model of carcinomatous Walker-256 cells in Wistar ratsObjective:To study the therapeutic effect of Fe₃O₄ nanometer magnetic fluid hyperthermia under an alternating magnetic field on subcutaneous tumor model of carcinomatous Walker-256 cells in Wistar rats in vivo.Methods:After subcutaneous tumor model of carcinomatous Walker-256 cells in Wistar rats was established,Fe₃O₄ nanometer magnetic fluid was injected into tumor tissues.Then,these Wistar rats were placed under an alternating magnetic field for hyperthermia（1,2 or 3 times per group respectively） with temperature controlled at 50～55 centigrade.The ratio of volume inhibition and survival time to subcutaneous tumor model in Wistar rats with Fe₃O₄ magnetic fluid hyperthermia were measured in contrast to control group s.And then,the pathologic changes and calefactive effect of Fe₃O₄ nanometer magnetic fluid under alternating magnetic field were observed.Results:1) Temperature in tumor tissues could reached to 50～55℃within 6 or 10 minutes,however,temperature in normal tissues was unchanged.2) From the volume-time curve,we could see that Fe₃O₄ nanometer magnetic fluid hyperthermia inhibited the tumor proliferation within two weeks,which was remarkable in MFH3 and MFH2 group than that in MFH1 and MF group in contrast to NS group s.The ratio of volume inhibition in MFH2 and MFH3 was 65%±3.5%、71.6±4.2% after hyperthermia at 14th day.3) The mean survival time of NS group was 35.50±5.59 days,MFH3 group:41.88±6.48 days,MFH2 group: 42.13±4.98 days,MFH1 group:39.00±5.30 days,MF group:36.06±6.57 days.The mean survival time of MFH3 and MFH2 group was longer than that of NS group,and there were statistical significance,q=-6.375, -6.625;P=0.003,0.002,however,there were no statistical significance between MFH1 and MF group with NS group,q=-3.500,-0.563;P =0.093,0.785.4) The tumor surface of NS and MF group was smooth,a few haemorrhage and small necrotic areas.The cancer cells were thick and fast,big nucleus and obvious nucleolus,and nucleus split stage could be seen easily.In the hyperthermia groups,the tumor surface was accidented and eschar.There were obvious haemorrhage,big necrotic areas and red-dyed remnant without structure,or cavity,and some nucleus shrank,split and dissolved.These manifests in MFH3 and MFH2 were obvious than those in NS and MF group.Conclusion:1) Nanometer magnetic fluid hyperthermia can ideally heat target tissues,however,temperature in normal tissues is unchanged, which is so-called "comformal hyperthermia".2) Fe₃O₄ nanometer magnetic fluid hyperthermia can inhibit the proliferation,prolong survival and promote apoptosis of subcutaneous tumor model of carcinomatous Walker-256 cells in Wistar rats.3) Animal experiments manifest that Fe₃O₄ nanometer magnetic fluid hyperthermia effect is relative to hyperthermia times and energy.4) From the above,we can draw a conclusion that Fe₃O₄ nanometer magnetic fluid hyperthermia may also act as a therapeutic tool for other solid tumors.CHAPTER THREE:Study on the angiogenesis effect of Fe₃O₄ nanometer magnetic fluid hyperthermia on subcutaneous tumor model of carcinomatous Walker-256 cells in Wistar ratsObjective:To investigate the angiogenesis effect of Fe₃O₄ nanometer magnetic fluid hyperthermia under an alternating electromagnetic field on subcutaneous tumor model of carcinomatous Walker-256 cells in Wistar rats in vivo.Methods:After subcutaneous tumor model of carcinomatous Walker-256 cells in Wistar rats was established,Fe₃O₄ nanometer magnetic fluid was injected into tumor tissues.Then,these Wistar rats were placed under an alternating magnetic field for hyperthermia（1,2 or 3 times per group respectively,I.e.MFH1,MFH2,MFH3） with temperature controlled at 50～55 centigrade.Reverse transcription-polymerase chain reaction and immunohistochemistry were used to analyze the vascular endothelial cell growth factor（VEGF） and receptor（Flk-1,Flt-1） mRNA,VEGF and microvessel density（MVD） among experiment groups and contrast group from specimens of subcutaneous tumor model in Wistar rats.Biological professional image analysis software（imagepro-plus 6.0） was used to analyze VEGF staining intensity（studied with the integrated optical density） and microvessel counting.At last,the correlation of VEGF and MVD was also analyzed.Results:1) The integrated optical density（IOD） values of VEGF dyed-cell and the numbers of MVD dyed-mirovessel in MFH3 and MFH2 groups with immunohistochemistry were lower than those in NS group, and the difference had statistical significance（p=0.000,0.001;0.000, 0.001）.However,IOD values and the numbers of MVD in MFH1 and MF groups were not different from those in NS group,and statistical significance was not found（p=0.675,0.999;0.332,0.994）.2) The correlation of VEGF and MVD was also analyzed,and statistical significance could be found,I.e.r=0.898,p=0.000.3) Fe₃O₄ nanometer magnetic fluid hyperthermia under an alternating electro-magnetic field could down-regulated VEGF mRNA and Flk-1 mRNA expression in MFH3 and MFH2 groups in contrast to those in NS group,and there were statistical significance,P=0.000,0.006;0.005,0.014.However,there were no statistical significance in MFH1 and MF groups,P=0.648,0.231; 0.994,0.158.4) Fe₃O₄ nanometer magnetic fluid hyperthermia could down-regulated Flt-1 mRNA expression in MFH3 groups in contrast to those in NS group,and there were statistical significance,P=0.009. However,there were no statistical significance in MFH1,MFH2 and MF groups,P=0.954,0.983,0.699.5) Flk-1 receptor was more sensitive to hyperthermia than Flt-1 receptor was at 50～55 centigrade,from the above,we could draw a conclusion that new-born vessel was more sensitive to hyperthermia than grown-up vessel was.Conclusion:1) Fe₃O₄ nanometer magnetic fluid hyperthermia under an alternating electro-magnetic field can down-regulate the expression of VEGF and VEGF receptors（Fit-1 and Flk-1）.Down-regulation of these factors may give rise to reduce of vessel number,which is confirmed by microvessel density count.2) New-born vessel is easier damaged than grown-up vessel to magnetic fluid hyperthermia.It indicates that the reduce of vessel number in tumor tissues mainly attribute to reduce of new-born vessel number.In other words,magnetic fluid hyperthermia inhibits new-born vessels.Indirect effect of magnetic fluid hyperthermia may be a more significant mechanism in tumor therapy.更多还原