【作者】 欧阳伟炜；

【导师】 唐劲天； 廖遇平；

【作者基本信息】 中南大学 ， 肿瘤学， 2010， 博士

【摘要】 研究背景恶性肿瘤严重威胁人类的健康,现有的放射治疗、化学治疗有较严重的副作用,手术会造成器官功能的损害。肿瘤热疗已成为继手术、放疗、化疗和生物免疫治疗之后的又一治疗肿瘤的重要手段。热疗能有效杀伤恶性肿瘤细胞,延长患者的生存时间,提高患者的生活质量,而且与放疗、化疗产生协同互补作用,增加患者对放疗和化疗的敏感性,同时又能减轻放疗、化疗的副作用,因而被国际医学界称为“绿色疗法”。过去热疗受技术所限加热温度不高,加热不均匀,治疗效果差异大。肿瘤的磁感应热疗把磁热介质导入肿瘤,其能将热能集中到肿瘤部位,实现所谓“适形热疗”同时使正常组织免受损伤,具有升温时间短,热分布均匀和微创等特点。热籽具有居里点自动控温特性,提高了加温治疗的安全性。在热籽加温治疗中我们首次利用大鼠Walker-256乳腺癌细胞株建立移植于大鼠乳腺部位的乳腺癌原位移植模型,利用此模型观察45℃常规高温治疗与50-55℃热切除治疗对大鼠乳腺癌的杀伤效果及对免疫功能的影响。磁性介质中磁性纳米粒子可以携带抗癌药物及放射性物质,增加基因的转染效率等实现肿瘤的局部热化疗、热放疗及热疗与生物免疫治疗的结合。热疗能增加肿瘤细胞膜对化疗药的通透性,降低耐药性的产生,因而热化疗能产生协同增敏作用。甲氨蝶呤是叶酸类似物,通过干扰肿瘤的代谢发挥抗肿瘤作用,然而肿瘤细胞对其易产生耐药性,我们在磁性纳米粒子上用高分子聚乙烯亚胺修饰后,再共价结合化疗药甲氨蝶呤,该磁性纳米粒子在交变磁场下产热对肿瘤可以产生热疗作用,同时加热促进磁性粒子上甲氨蝶呤的释放,促进细胞对甲氨蝶呤的摄取,提高肿瘤治疗的疗效。甲氨蝶呤偶联的磁性纳米粒子粒径约10nm,呈超顺磁性。目前含甲氨蝶呤化疗药的超顺磁性纳米粒子热化疗治疗动物肿瘤实验还没有开展,我们用此磁流体研究热化疗对大鼠乳腺癌的治疗效果,为磁感应热化疗的可行性提供实验及理论依据。目的建立大鼠乳腺原位移植性乳腺癌模型观察肿瘤的生物学特性。方法分为2组：接种肿瘤观察组大鼠40只,正常组大鼠4只。接种肿瘤观察组在大鼠右前胸部乳腺处皮下接种Walker-256乳腺癌细胞株观察肿瘤的生长、生存期、病理学检查及死后解剖有无脏器转移。接种后2周及3周均随机处死2只大鼠取肿瘤组织并观察大鼠有无脏器转移。正常组大鼠予处死取乳腺组织。结果正常大鼠乳腺见较广泛的脂肪组织分布,乳腺呈单层上皮分布,未见明显的增生改变。接种肿瘤的乳腺组织可见肿瘤侵犯乳腺上皮后乳腺出现明显的上皮增生、核异型性改变、甚至癌变。大鼠肿瘤成瘤率80%,肿瘤呈圆形或椭圆形突出于体表,生存期(37.10±8.54)天。2周处死大鼠未见脏器肿瘤转移,3周处死大鼠见肺转移。大鼠59.3%(19/32)肺转移,12.5%(4/32)肝转移,6.25%(2/32)腋下淋巴结转移,6.25%(2/32)皮下转移等,未见明显的骨转移。结论大鼠肿瘤生长相对较快,与人类常见乳腺癌不相符合,与炎性乳癌相似,可作为恶性肿瘤基础研究的模型。目的评估45℃常规热疗与50℃-55℃热切除治疗对大鼠乳腺原位移植性乳腺癌的治疗效果及对免疫功能的影响。方法185只在右前胸部乳腺皮下接种Walker-256乳腺癌细胞的荷瘤Wistar大鼠进行随机分组实验：C组(空白对照组,37只),M组(磁场对照组,31只),T组(热籽对照组,31只),H组(加温治疗H1组43只,H2组43只)。H1组：45℃常规热疗,治疗时间30分钟；H2组：50℃-55℃热切除治疗,治疗10分钟。加温治疗后即刻、治疗后12h及24h每组均处死2只大鼠行常规病理检查,HSP70、Bax、Bcl-2、PCNA的免疫组化检测和凋亡检测。热疗后2周在C组、H1组与H2组每组处死5只大鼠检测外周血T淋巴细胞亚群及IL-2和IFN-γ水平。评估大鼠肿瘤体积及观察长期生存。结果可见肿瘤侵入乳腺组织,乳腺上皮增生、核异型性明显甚至癌变。热疗后H1组Bax蛋白表达增加Bcl-2表达下降,肿瘤细胞大量凋亡与H2组比较差异有显著性(P<0.05)。H2组肿瘤细胞见大片的坏死。H1组与H2组加温治疗后即刻PCNA表达开始减弱,治疗后12h及24h后只有少量残存的肿瘤细胞表达PCNA与C、M及T组比较差异有显著性(P<0.05)。H1组与H2组加温治疗后即刻HSP70的表达增加,治疗后12h及24h后残存的少量肿瘤细胞表达HSP70与C、M及T组比较差异有显著性(P<0.05)。热疗后2周肿瘤体积H1组、H2组与3个对照组比较差异有显著性(P<0.05)。H1组、H2组与3个对照组比较生存期明显延长(P<0.01),H2组比H1组长(P<0.05)。外周血T淋巴细胞亚群CD4+在H1组、H2组比C组高,差异有显著性(P<0.05),H1组比H2组高,差异有显著性(P<0.05)。CD4+/CD8+比值在H1组、H2组比C组高,差异有显著性(P<0.05),H1组比H2组高,差异也有显著性(P<0.05)。血清IL-2水平H1组、H2组比C组高,差异有显著性(P<0.05)。血清IFN-γ的水平H1组、H2组比C组高,差异有显著性(P<0.05),H2组比H1组高,差异有显著性(P<0.05)。结论45℃常规热疗与50℃-55℃热切除治疗对大鼠原位移植性乳腺癌的生长有一定的抑制作用,50℃-55℃热切除治疗大鼠的存活率高,两组均能提高机体的免疫功能。目的磁性纳米粒子在生物医学工程领域有较广泛的应用,如局部肿瘤的磁感应热疗、药物的传递及核磁共振成像及基因转染等。甲氨蝶呤(MTX)是叶酸类似物,通过干扰肿瘤的代谢发挥抗肿瘤作用,然而其毒副作用大,而且肿瘤细胞对其易产生耐药性,为了减少它的毒副作用,避免耐药性的产生,研究者往往采用将其与一些高分子载体偶联,然而这种方式仅能产生单一的化疗作用。本研究采用聚乙烯亚胺(PEI)修饰Fe3O4磁性纳米粒子作为热介质,将MTX共价结合在PEI修饰的磁性纳米粒子上,这样实现磁感应热疗及化疗的双重治疗作用,通过该磁性介质对荷瘤大鼠治疗效果的观察及对免疫功能的影响的研究,为磁感应热化疗的可行性提供实验及理论依据。方法自制PEI修饰的Fe3O4磁性纳米粒子,粒子呈超顺磁性,粒径约10nm。MTX共价修饰的磁性粒子也呈超顺磁性,粒径约10nm。在大鼠右前胸部乳腺处皮下接种Walker-256乳腺癌细胞,7-10天后大鼠长出1.5-2cm的肿瘤进入实验,在肿瘤内注射1/2肿瘤体积量的300mg/ml的含PEI或MTX化疗药的磁流体,24h后在肿瘤周围注射1ml的磁流体30min后进入交变磁场(300KHz)中升温治疗。予磁流体体外测温及体内温度监测。分为以下十二组：47℃热化疗组(47TC)：24只,含MTX的磁流体在交变磁场中升温到47℃,治疗时间30min；47℃热疗组(47T)：24只,PEI修饰的磁流体在相同磁场中升温到47℃,治疗时间30min；单次42℃热化疗组(单次42TC)：21只,含MTX磁流体在磁场中升温到42℃,治疗时间60min；单次42℃热疗组(单次42T)：21只,PEI修饰的磁流体在磁场中升温到42℃,治疗时间60min；多次42℃热化疗组(多次42TC)：12只,含MTX磁流体在磁场中升温到42℃,治疗时间60min,治疗间隔72h,治疗2-5次；多次42℃热疗组(多次42T)：12只,PEI修饰的磁流体在磁场中升温到42℃,治疗时间60min,治疗间隔72h,治疗2-5次；磁流体化疗药物对照组(MFC)：16只,注射含MTX化疗药的磁流体不进入磁场升温,治疗后24 h处死2只大鼠；空白对照组(C)：21只,接种肿瘤大鼠不做任何处理；磁场对照组(M)：12只,在交变磁场中辐照30min；磁流体对照组(MF)：12只,注射PEI修饰的磁流体不进入磁场治疗；MTX化疗药组(MTX)：16只,在肿瘤内注射20mg/kg的MTX化疗药,治疗后24h处死2只大鼠；正常组(Normal):10只,不接种肿瘤观察生存情况。治疗后即刻除正常组、MFC、MTX组外每组处死2只行病理检查。治疗后12h 47℃热化疗组、47℃热疗组、单次42℃热化疗组、单次42℃热疗组、空白对照组每组处死2只,24h每组处死3只行病理检查、电镜检查、PCNA、HSP70免疫组织化学检测及血常规、生化检查。治疗后2周47TC组、47T组、单次42TC组、单次42T组、MFC、MTX、C组及正常组大鼠每组处死4只取外周血做IL-2、IFN-γ、IL-4免疫功能检测和血常规、生化检查,其余大鼠观察生存情况。结果磁流体升温较好,治疗后肿瘤组织内见大量的磁性纳米粒子分布,热化疗及热疗组肿瘤组织出现凋亡、坏死改变,以47℃治疗组明显。治疗后24h HSP70、PCNA的表达在47TC及47T组明显降低与单次42TC、单次42T、MFC、MTX、C组比较有显著性差异(P<0.05)。治疗后肿瘤体积47TC组、47T组、多次42T组与单次42TC、单次42T、MFC、M、MF、MTX、C组比较差异有统计学意义(P<0.05),多次42TC组与单次42T、MFC、M、MF、MTX、C组比较差异有统计学意义(P<0.05)。长期生存观察除正常组外,47TC组、47T组、多次42TC组、多次42T组与C、M、MF、MTX组比较差异有显著性(P<0.05),47℃热化疗组存活率84.6%(11/13)。47TC及47T组外周血血清IL-2水平比单次42T及C组高,差异有显著性(P<0.05),与正常组比较无显著性差异。IFN-γ水平在47TC组、47T组、单次42TC组比C组高,差异有显著性(P<0.05)。IL-4水平在各组比C组低,差异有显著性(P<0.05)。磁流体热化疗后24h白细胞降低,部分大鼠转氨酶升高。治疗后2周血象及肝、肾功能无明显异常。结论热化疗可能比单纯的热疗及化疗疗效好,47℃热化疗存活率最高。47℃热化疗/热疗与多次42℃热化疗、多次42℃热疗一定程度上抑制了肿瘤的生长,47℃热化疗/热疗较单次的42℃热化疗/热疗激活了机体免疫功能。MTX修饰的磁流体无明显的毒副作用值得进一步研究。更多还原

【Abstract】 Backgrounds:Malignant carcinoma has become a serious threaten to human health nowadays. Traditional methods such as radiotherapy, chemotherapy and surgery have unbearable side effects, surgery can cause organ damage. Following surgery, radiotherapy, chemotherapy and immune therapy, hyperthermia has become another important method to treat cancer. The property of the thermotherapy is to directly kill the tumor cells; therefore it can prolong the survival time and improve the quality of life of patients. When it coupled with other treatment methods including radiotherapy and chemotherapy, it can significantly enhance the sensitivity and reduce the side effects of other treatment methods. Thus the thermotherapy is viewed as "green therapy" by the international medical community. According to traditional technology, the heating temperature is not high, uneven heating, which induce instability of therapeutic effect. The magnetic induction hyperthermia treatment can concentrate energy to the regional mass by magnetic medium, which is also called "comformal thermotherapy". As a result, there are many advantages including protecting normal tissue, shorter heating time, better heat disposition and less invasive operation. Thermoseeds has characteristic of automatic control of the temperature with Curie point, so it improves the safety of the thermetherapy. We firstly set the model of the mammary orthotopic transplantation tumors in rats by Walker-256 strain in the thermoseed hyperthermia treatment. We observe the effect conventional high-temperature in 45℃and 50℃-55℃thermal ablation therapy on rat anti-breast cancer and its influence on immune function. Magnetic nanoparticles media can carry anticancer drugs and radioactive substances and increase gene transfection efficiency, which were called thermo-chemotherapy, thermo-radiotherapy, and thermo-genetics respectively. Hyperthermia can increase the tumor cell membrane permeability to chemotherapeutic drugs and reduce drug resistance so that thermotherapy and chemotherapy can produce synergistic sensitizing effect. Methotrexate (MTX) is a folic acid analogue, whose anti tumor effects is obtained by interfering with the metabolism of tumor, but it is easy to tumor cell resistance. We use magnetic nanoparticles modified with polyethylene imine, and then covalent chemotherapy MTX, the magnetic nanoparticles in alternating magnetic field can generate heat on the tumor mass, while increase release MTX, promotes cell uptake of MTX to enhance efficacy of cancer treatment. MTX conjugated magnetic nano-particle size of about 10nm, showed superparamagnetic. Currently, MTX superparamagnetic nanoparticles cancer thermochemotherapy on animal experiments have not been reported, and we use the magnetic fluid on rat breast cancer treatment which aimed to research feasibility of magnetic induction to provide the experimental and theoretical basis. Objective To establish transplantable transplantable metastasis models of breast Cancer and observe their biological characteristics.Methods Two groups contains 40 rats in the inoculated group and 4 normal rats in the control group. The rats were inoculated by Walker-256 breast cancer cells lines in the rats’right mammary gland, then we observed the tumor growth, survival, pathological examination and the autopsy with or without visceral metastasis etc, which could help understand their biological characteristics. Two weeks and 3 weeks later, 2 rats were randomly killed, the tumor tissue was resected and were observed organ whether or not metastasis. Normal rats were sacrificed to the breast tissue.Results In normal rat, the mammary reveals the normal distribution of adipose tissue, mammary is epithelial monolayer without any change of the proliferation. The glands inoculated with cancer cells was found the solid tumor were round or oval-shaped protruding surface, and with epithelial hyperplasia, nuclear atypia change, and even carcinogenesis. Tumor formation rate is 80%, and the survival time is (37.10±8.54) days. No metastasis occurred in the 2nd week, and the metastasis happened in the 3th week,59.3%(19/32) of rats was occurred metastasis in the lung,12.5%(4/32) liver,6.25%(2/32) axillary lymph node,6.25%(2/32) subcutaneous transfer, bone metastasis was none. Conclusion The tumor grows relative rapid, and it was not similar to the common breast cancer of human, but to the inflammatory breast cancer, which would be used as a cancer model for basic research.Objective The study was to evaluate the effect of thermoseed inductive heating on mammary orthotopic transplantation tumors and immunologic function in rats.Methods Walker-256 tumor cells were inoculated subcutaneously into the mammary glands of Wistar rats. Rats were allocated to five groups:(I) C group (control group,37 rats); (II) M group (magnetic field group,31 rats); (III) T group (thermoseed control group,31 rats); (IV) H1 group (Hyperthermia treatment:45℃for 30 min,43 rats); (V) H2 group (Hyperthermia treatment:50℃-55℃for 10 min,43 rats). Immediately, 12 h and 24 h after hyperthermia, two rats were sacrificed in each group for pathological examination. Immunohistochemical method was used to examine the expression of PCNA, HSP70, Bax, and Bcl-2 and detect apoptosis. T lymphocyte subgroups, IL-2 and IFN-γlevels were measured in C, H1 and H2 groups after two weeks hyperthermia. Tumor volume was measured and long-term survival was observed.Results Breast tissue was infiltrated by the tumor, with the epithelial hyperplasia, marked nuclear atypia and even carcinomatous. After the hyperthermia, the Bax protein expression increased (P<0.05) amd Bcl-2 expression decreased (P<0.05) in H1 group. And the apoptosis was statistically different compared with H2 group (P<0.05). The expression of PCNA began to decrease immediately after the heating in group H1 and H2 and few residual tumor cells expressed PCNA after treatment of 12h or 24h, which was significant different from the C, M and T group (P<0.05). The expression of HSP70 began to increase immediately after the heating in group H1 and H2, few residual tumor cells expressed HSP70 after 12h and 24h’s treatment, which was significantly different from the C, M and T group(P<0.05). And the difference of the tumor volume between the H1, H2 and the other 3 controlled groups was significant (P<0.05). The survival time of the H1 Group and H2 group was longer than the other groups (P<0.01), which of H2 group was longer than H1 (P<0.05). CD4+T lymphocyte subsets of peripheral blood in the H1 group, H2 group was statistically higher than in the C group (P<0.05), which in H1 group was statistically higher than the H2 group (P<0.05). CD4+/CD8+ ratio in H1 group, H2 group was higher than in the C group (P<0.05), which in H1 group was higher than in H2 group (P<0.05). Serum levels of IL-2 of H1 group, H2 group was higher than in C group (P<0.05). Serum levels of IFN-y of H1 group, H2 was higher than in C group (P<0.05), which in H2 group was higher than in H, group (P<0.05).Conclusion Both general hyperthermia in 45℃and thermal ablation therapy in 50℃-55℃on breast cancer orthotopic transplantation inhibited the growth, thermal ablation treatment in 50℃55℃is of the high survival rate of rats, and immune function was enhanced in both groups.Objective Magnetic nanoparticles has been widely applied in the field of the biomedical engineering, such as local tumor magnetic hyperthermia, drug delivery, magnetic resonance imaging and gene transfer, etc. Methotrexate (MTX) is a folic acid analogue, which has antitumor effect by interfering with the metabolism.But MTX was own seriously toxic side effects, and it is easy to tumor cell resistance. To reduce its side effects and avoid drug resistance, researchers often use their carrier with a number of polymer coupling, but this approach can only produce a single effect of chemotherapy. In this study, MTX covalent coupled with polyethylenimine (PEI) which was the magnetic induction thermochemotherapy achieves the dual therapeutic effect. We observe the effect to tumor-bearing rats and impact on immune function through the magnetic induction thermochemotherapy and explore feasibility of experiments and theoretical basis.Methods The Fe3O4 magnetic nanoparticles modified by PEI were superparamagnetic, with the size of about lOnm. The magnetic particles covalent modified by MTX was also superparamagnetic and with the size of 10nm. We inoculated the walker-256 breast cancer cells to the right mammary of the rats, the ones whose tumor size grew to 1.5-2cm after the inoculation were selected in to the experiment. The tumor was injected with the 300mg/ml magnetic fluid with the PEI or MTX, which was a half volume of the tumor.24h later, 1ml magnetic fluids were inoculated at the peripheral area of tumor. After 30 minutes, rats were placed in the alternating magnetic fields (300 kHz) to receive hyperthermia. And then we monitor the temperature in vitro and in vivo. The rats were divided into 12 groups:47℃thermochemotherapy group (47TC):24 rats were treated with MTX modified magnetic fluid in an alternating magnetic field in the temperature to 47℃, the treatment time is 30 min; 47℃hyperthermia group (47T):24 rats were with PEI modified magnetic fluid in the same temperature to 47℃, the treatment time is 30 min; single 42℃thermochemotherapy (single 42TC):21 rats with MTX modified magnetic fluid in magnetic field heating to 42℃, the treatment time is 60 min; Single 42℃hyperthermia group (single 42T): 21 rats, PEI modified magnetic fluid in magnetic field heating to 42℃, treatment time is 60min; repeatedly at 42℃chemotherapy group (repeated 42TC):12, with MTX modified magnetic fluid in magnetic field heating to 42℃, the treatment time is 60 min, interval of 72h,2-5 times; Repeated 42℃hyperthermia group (repeated 42T):12 rats, PEI modified magnetic fluid in magnetic field heating to 42℃, the treatment time is 60 min, and interval of 72h,2-5 times; magnetic fluid chemotherapy group (MFC):16 rats injected with MTX modified magnetic fluid did not enter the magnetic field, After 24 h two rats were killed; blank control group (C):21, tumor growth in rats without any treatment; magnetic control group (M):12, rats were exposured 30min in the alternating magnetic field; magnetic fluid control group (MF):12 rats injected with PEI modified magnetic fluid did not enter the magnetic field; MTX chemotherapy group (MTX):16 rats were intratumoral injected with 20mg/kg of MTX; normal group (Normal):10 rats, observe the survival without tumor inoculation. Immediately after treatment in each group except normal group, MFC group, MTX group were killed for the pathological examination. After 12h,47TC,47T, single 42TC, single 42T, C group were sacrificed 2 rats each. After 24h,3 rats were killed for pathological examination, electron microscopy, PCNA, HSP70 immunohistochemistry and routine blood and biochemical examination.2 weeks later,4 rats were killed in 47TC,47T, single 42TC, single 42T, MFC, MTX, C group and the normal group for detect the IL-2, IFN-γ, IL-4 and routine blood and biochemical examination, the remaining rats were observed survival time.Results Magnetic fluid in vivo, in vitro temperature well. After treatment, tumor tissues revealed a large number of magnetic nano-particle distribution. Thermochemotherapy and hyperthermia induced apoptosis and necrosis, the 47℃treatment group was of the most distinction.24 h later, HSP70, PCNA expression in 47TC and 47T were significantly lower than that of single 42TC, single 42T, MFC, MTX, C groups (P<0.05). Two weeks after treatment, tumor volume in 47TC,47T, and repeated 42T group was significantly small than that in single 42TC, single 42T, MFC, M, MF, MTX, C group (P<0.05), and repeated 42TC group was also small than that in single 42T, MFC, M, MF, MTX, C group (P<0.05). The survival time of the 47TC,47T, repeated 42TC, and repeated 42T were longer than M, MF, MTX, C group (P <0.05).47TC group survival rate was 84.6%(11/13). The serum levels of IL-2 with 47TC and 47T were significantly higher than of single 42T and C (P<0.05), and there were no difference between 47TC,47T group and normal groups. IFN-γlevels in 47TC,47T, and single 42TC were significantly higher than of C group (P<0.05). IL-4 levels in C group were significantly higher than of other groups (P<0.05). Methotrexate modified Magnetic fluid reduced the number of white blood cells in 24h after thermochemotherapy, and transaminase of some rats were elevated. Two weeks after treatment, routine blood, liver and renal function were normal.Conclusion Thermochemotherapy is more effective than thermotherapy or chemotherapy single, and 47℃thermochemotherapy is the group of the highest survival rates.47℃thermochemotherapy/ hyperthermia and repeated 42℃thermochemotherapy/hyperthermia can inhibite tumor growth to a certain extent, and 47℃thermochemotherapy /hyperthermia can activate immune function than single 42℃thermochemotherapy/hyperthermia. MTX modified magnetic fluid has not serious toxicity, which is worth to further study.更多还原