纳米磁流体热疗治疗人舌癌裸鼠异种移植瘤的实验研究

发布时间：2018-05-20 13:40

  本文选题：纳米磁流体 + 磁流体热疗　； 参考：《山东大学》2014年硕士论文

【摘要】：口腔鳞状细胞癌(oral squamous cell carcinoma, OSSC)是口腔颌面部最常见的恶性肿瘤,来源于皮肤和黏膜的复层鳞状上皮细胞。而在口腔鳞状细胞癌中,最常见的类型之一就是舌鳞状细胞癌。近年来口腔鳞状细胞癌的发病率呈上升趋势,发病年龄也趋于年轻化。寻找积极有效的治疗途径成为一项十分迫切的重要任务。手术疗法、化学疗法、放射疗法依然是目前最主要的三种治疗舌鳞状细胞癌的手段,但手术治疗的损伤和风险以及放化疗的巨大毒副作用严重影响了肿瘤患者的生存质量和存活率。因此,研究和开发具有靶向性的、更加安全、更加有效、低副作用的、无创或微创的舌鳞状细胞癌治疗新方案势在必行。 利用肿瘤组织较正常组织对高温更加敏感的原理,将肿瘤组织加热到43℃以上,肿瘤细胞较正常细胞不能耐受持续高温,从而发生不同程度的凋亡或坏死,这种治疗恶性肿瘤的方法称热疗(Hyperthermia)。近年来,纳米技术的迅速发展使其成为在医学领域具有挑战性的创新。磁流体热疗(Magnetic fluid hyperthermia, MFH)是将经过包裹修饰或添加了某种抗体的磁流体通过动静脉或直接注射到达肿瘤内部,磁流体被肿瘤细胞吞噬或沉积在细胞之间,在交变磁场(Alternating magnetic field, AMF)下,磁性纳米颗粒通过磁矢量旋转和颗粒本身的物理旋转,即通过奈尔松弛和布朗弛豫机制升温,从而杀死肿瘤细胞,而周围正常组织升温不明显,具有高度的靶向性。磁流体热疗技术的出现解决了热疗中病灶准确定位的问题,在肿瘤热疗方面取得较好的疗效。磁流体热疗是一项对深部组织热疗的全新方法,可以通过调节磁流体的浓度或磁场强度来得到我们实验中想要的热疗温度,本实验是通过调节磁流体浓度来实现的；局部热疗法可分为温热疗法(42-46℃)、高热疗法(46-70℃)、热切除(70℃以上)。与传统的热疗技术如红外热疗、超声热疗、微波热疗和射频消融、电磁波热疗相比,MFH技术具有可治疗深层肿瘤、副作用小、微创甚至无创的特点,且靶向性好,且肿瘤部位单次注射纳米磁流体,可以对病人进行重复多次热疗。 裸鼠免疫功能缺陷,在一定条件下,不排斥来自异种动物的组织移植；被移植肿瘤仍保持原有组织学结构和生物学特性；同时,几乎很少见移植肿瘤发生转移。这些条件是成功建立人舌癌肿瘤模型的基础,也是本实验的重要保证；且裸鼠腋窝皮下肿瘤模型,肿瘤组织生长表浅易从体外扪及,肿瘤血液供应丰富,肿瘤生长较大,可以有利于观察磁流体热疗治疗对肿瘤体积变化的影响。纳米粒子的磁流体热疗的应用已被证明在几种动物模型中能够抑制肿瘤的生长,然而,使用磁流体热疗在体内治疗舌癌的可行性是不确切的,其机制也尚不清楚。在目前研究中,本课题旨在建立裸鼠腋窝皮下舌癌动物模型,瘤内注射纳米磁流体后,在交变磁场下热疗治疗,观察磁流体热疗对肿瘤生长的影响,进一步验证纳米磁流体热疗治疗恶性肿瘤的可行性及临床应用价值。 目的： 检测二氧化硅包裹的Mn0.4Zn0.6Fe2O4纳米磁流体在交变磁场下的体内外升温性能,建立Tca8113人舌鳞癌细胞裸鼠皮下移植瘤模型,探讨Mn0.4Zn0.6Fe2O4纳米磁流体局部热疗治疗裸鼠Tca8113舌癌模型的可行性及其对瘤体生长的影响。 方法： 1.Tca8113细胞的培养：复苏舌癌细胞株Tca8113细胞,RPMI160培养基体外培养,观察Tca8113细胞的形态及增值活性。 2.将Mn0.4Zn0.6Fe2O4纳米磁流体进行透射电子显微镜检测、磁滞回线检测以及体外不同磁场电流强度下的升温实验检测。 3.人舌癌Tca8113细胞对Mn0.4Zn0.6Fe2O4纳米磁颗粒的吞噬实验：取处于对数生长期的Tca8113细胞,与加入含有二氧化硅包裹的锰锌铁氧体纳米颗粒的培养基共培养24h,普鲁士蓝染色,倒置显微镜下观察细胞对纳米颗粒的吞噬情况。 4.Tca8113细胞毒性检测及细胞形态学观察：将含有不同浓度纳米粒子浸提液的RPMI-1640培养基与Tca8113细胞共同培养,观察细胞形态学的改变,检测细胞增殖活性的改变,以及对细胞的毒性检测。 5.体内试验：随机选择BALB/c-nu雄性裸鼠27只,人舌鳞癌细胞Tca8113腋窝皮下注射建立裸鼠肿瘤模型。将接种10天的荷瘤鼠随机分为5组：即空白对照组(A组)、磁流体对照组(B组)、磁场对照组(C组)、磁流体热疗1组(D组)、磁流体热疗2组(E组)。磁流体热疗1组和磁流体热疗2组直接瘤内注射磁流体24h后暴露于交变磁场,用热敏电偶温度传感器测量交变磁场下瘤体中心、瘤体边缘及肛周的温度变化。当实验组肿瘤中心区温度稳定在43℃后,再持续热疗30min。磁流体热疗2组在5～7后再重复加热一次。磁场对照组加热时间及加热条件与磁流体热疗组完全相同。而空白对照组不经任何处理,磁流体对照组只瘤内注射纳米磁流体未经磁场加热。热疗治疗结束后24h,各组随机选择1只裸鼠,麻醉注射过量处死,迅速取出肿瘤组织、周围肌肉组织、心脏、肺、肝脏、脾脏和肾脏,肉眼大体观察组织的病理改变。剖开即刻用4%甲醛固定,石蜡包埋切片,HE和普鲁士蓝染色,光学显微镜下观察肿瘤组织病理学变化及重要脏器内有无纳米磁流体残留。九周后处死所有实验组裸鼠,测量瘤体体积,计算肿瘤体积抑制率,肉眼大体观察标本并进行组织病理学检查。 6.统计学分析：统计学分析采用SPSS20.0软件包处理。计量数据采用均数±标准差(x±s)表示,吸光度值、肿瘤体积、肿瘤最长径和各组温度采用完全随机设计资料的单因素方差分析,进一步采用L.S.D.法做组间、组内两两之间比较。P0.05为差异具有统计学意义。 结果： 1.Tca8113细胞的培养：Tca8113细胞形态符合人舌鳞癌细胞形态特征,在倒置显微镜下观察可发现,单个细胞呈长梭形或多角形,呈集落性生长,增殖较快,约第二天开始进入对数生长期,第四天进入平台期。 2.在透射电子显微镜下观察拍照发现纳米粒子颗粒在水溶液中分散较好,粒径约10nm,没有聚集现象发生；通过磁滞回线检测,锰锌铁氧体纳米粒子具有超顺磁性。体外磁流体升温实验检测发现纳米磁流体升温速率随磁流体浓度增大而增大,在电流为50A和磁场频率为237KHZ时,磁流体可在20min内快速上升至80℃变缓慢最后维持在83℃不上升。 3.Tca8113细胞与Mn0.4Zn0.6Fe2O4颗粒共培养24h后,倒置显微镜观察发现,普鲁士蓝染色法,Tca8113细胞内有呈蓝色改变的氧化铁颗粒,而空白对照组无蓝色颗粒。 4.25%、50%、75%浸提液组分别与Tca8113细胞共培养,细胞数量随着培养时间的延长细胞数量逐渐增多,细胞形态未见明显变化,无细胞毒性；100%浸提液组生长缓慢,可见少量细胞核固缩,表现为轻微的细胞毒性。随着浸提液浓度的增高,MTT检测细胞的增殖活性明显降低。 5.裸鼠接种10d时腋窝皮下瘤体直径约0.8cm-lcm,裸鼠成瘤率100%。随机分为5组,热疗前各组裸鼠瘤体体积之间比较,差异无统计学意义(P0.05)。磁流体热疗组肿瘤中心温度可相对稳定在43℃,瘤体边缘区温度比瘤体中心区温度低2-3℃约40℃,直肠几乎不升温。磁场对照组无升温现象,肿瘤中心温度与体温相同。空白对照组和磁流体对照组裸鼠肿瘤中心温度稳定在30.8℃,也无升温现象。磁流体热疗1组和磁流体热疗2组肿瘤中心区、肿瘤边缘区温度与空白对照组进行比较,差异具有统计学意义(P0.05)。磁流体热疗1组与磁流体热疗2组之间进行比较,差异无统计学意义(P0.05)。磁流体热疗组肿瘤中心区与肿瘤边缘区之间比较,差异具有统计学意义(P0.05)。所有实验裸鼠均能耐受热疗。各组裸鼠经过热疗治疗后瘤体体积均呈一定程度的增大趋势。空白对照组和磁场对照组增大趋势较为明显,磁流体对照组增长趋势稍低。磁场对照组、磁流体对照组、空白对照组即三对照组间肿瘤体积进行比较,差异无统计学意义(P0.05)；磁流体热疗组裸鼠处理后瘤体体积6周内即种瘤后第2、3、4、5周肿瘤体积与对照组之间进行比较,差异具有统计学意义(P0.05),尤其是磁流体热疗2组肿瘤体积与各对照组之间比较差异更为显著(P0.01)；两磁流体热疗组在种瘤后第3、4、5周肿瘤体积比较差异亦具有统计学意义(P0.05),但6周之后由于热疗瘤体周边残留的肿瘤组织细胞继续增长,6周后各组肿瘤体积之间进行比较,差异无统计学意义(P0.05)。磁流体热疗1组和磁流体热疗2组瘤内注射磁流体热疗治疗后,肿瘤生长受到抑制,磁流体热疗2组抑制作用更为明显,磁流体热疗1组与磁流体热疗2组在种瘤后2-5周肿瘤体积抑制率分别为23.80%～41.81%和34.92%～56.76%。各组心脏、肺、肝脏、肾脏和脾脏等重要器官未见有肿瘤细胞的转移,也未见纳米磁流体的残留,无明显异常改变。 结论： 1.Mn0.4Zn0.6Fe2O4纳米磁流体水溶性好,具有磁性；Mn0.4Zn0.6Fe2O4纳米磁流体在体内外均具有良好升温性能的特性,在体内可使肿瘤区域获得比较均匀分布的43℃热疗治疗温度,而其余正常组织不升温。 2.人舌Tca8113细胞可吞噬Mn0.4Zn0.6Fe2O4纳米颗粒。 3.Mn0.4Zn0.6Fe2O4磁流体对细胞无明显细胞毒性,毒性与磁流体浓度密切相关；且磁流体对舌癌Tca8113细胞形态没有明显的影响。 4.成功构建舌癌动物模型,组织病理学观察确定为Tca8113鳞状细胞癌。43℃磁流体热疗可导致肿瘤组织明显坏死,显著抑制肿瘤生长；同样,磁流体热疗可促进瘤内纳米磁流体的分散,实现热疗的周边效应。磁流体热疗对肿瘤组织细胞的杀伤作用与热疗频率有关。一次性瘤内注射纳米磁流体后,磁流体介导的43℃热疗治疗裸鼠Tca8113皮下移植瘤安全、有效、可行、靶向性好,低副作用,且可进行重复热疗,治疗肿瘤具有潜在的临床应有价值。
[Abstract]:Oral squamous cell carcinoma (OSSC) is the most common malignant tumor in the oral and maxillofacial region. It comes from the complex squamous cells of the skin and mucosa. In oral squamous cell carcinoma, one of the most common types of oral squamous cell carcinoma is the squamous cell carcinoma of the tongue. Age is also becoming younger. Finding active and effective ways of treatment has become an urgent and important task. Surgery, chemotherapy, and radiotherapy are still the three most important methods for the treatment of squamous cell carcinoma of the tongue, but the damage and risk of surgical treatment and the huge toxic and side effects of radiotherapy and chemotherapy have seriously affected the cancer patients. Therefore, it is imperative to study and develop a new approach to the treatment of squamous cell carcinoma of the tongue, which is targeted, safer, more effective, and less side effects, without invasive or minimally invasive tongue squamous cell carcinoma.
Using the principle that tumor tissue is more sensitive to high temperature than normal tissue, the tumor tissue is heated to more than 43 degrees centigrade, and the tumor cells are not tolerable to the normal cells, which can not tolerate the continuous high temperature, thus the different degrees of apoptosis or necrosis occur. The method of treating malignant tumor is called Hyperthermia. In recent years, the rapid development of nanotechnology has made it make it For a challenging innovation in the medical field, Magnetic fluid hyperthermia (MFH) is a magnetic fluid that has modified or added some kind of antibody to the inside of the tumor via an intravenous or direct injection. The magnetic fluid is phagocytic or deposited in the cell between the tumor cells, in the Alternating magnetic field (Alternating magnetic field, AMF) the magnetic nanoparticles rotate through the magnetic vector and the physical rotation of the particles themselves, that is, the tumor cells are killed by Nair relaxation and the Brown relaxation mechanism, and the normal tissue around the normal tissue is not obviously heated and highly targeted. The emergence of magnetic fluid thermotherapy technology solves the problem of the accurate location of the focus in the hyperthermia, and it is swollen in the swelling. The magnetic fluid thermotherapy is a new method for the hyperthermia of the deep tissue. It can be obtained by regulating the concentration of magnetic fluid or the intensity of the magnetic field. This experiment is achieved by adjusting the concentration of magnetic fluid; the local thermotherapy can be divided into thermotherapy (42-46), high Hyperthermia (46-70 C), hot excision (above 70 degrees C). Compared with the traditional thermotherapy, such as infrared thermotherapy, ultrasound thermotherapy, microwave hyperthermia and radiofrequency ablation, electromagnetic wave thermotherapy, MFH technology has the characteristics of treatment of deep tumor, small side effect, minimally invasive and even noninvasive, and the tumor site is injected with nanoscale magnetic fluid, which can be used for patients. Repeated hyperthermia were repeated.
The immune deficiency of nude mice, under certain conditions, does not exclude tissue transplantation from different species of animals; the transplanted tumor remains the original histologic structure and biological characteristics; at the same time, the metastasis of the transplanted tumor is almost rare. These conditions are the basis of the successful establishment of the tumor model of the cancer of the tongue, and also an important guarantee for this experiment. The tumor tissue growth table is easy to be palpable in vitro, rich in blood supply and large growth of tumor, which can help observe the effect of magnetic fluid thermotherapy on tumor volume change. The application of magnetic fluid thermotherapy for nanoparticles has been proved to be able to inhibit tumor growth in several animal models, however, however, The feasibility of using magnetic fluid thermotherapy in the treatment of tongue cancer in vivo is not accurate and its mechanism is still unclear. In the present study, the aim of this study is to establish an animal model of subcutaneous tongue cancer in nude mice. After intratumoral injection of magnetic fluid, the effect of thermotherapy on the growth of tumor is observed under the alternating magnetic field and the effect of magnetic fluid thermotherapy on tumor growth is observed. The feasibility and clinical application value of M magnetic fluid hyperthermia in the treatment of malignant tumors.
Objective:
To detect the heating performance of Mn0.4Zn0.6Fe2O4 nano magnetic fluid encapsulated by silica in the alternating magnetic field in vivo and in vitro, the model of subcutaneous transplantation of Tca8113 human tongue squamous cell carcinoma cells in nude mice was established, and the feasibility of Mn0.4Zn0.6Fe2O4 nano magnetic fluid local thermotherapy in the treatment of Tca8113 tongue cancer model in nude mice and its effect on the growth of the tumor were discussed.
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