【作者】 杜益群；

【导师】 张东生；

【作者基本信息】 东南大学 ， 病理学与病理生理学， 2006， 硕士

【摘要】 [目的]①研究Fe₃O₄磁性纳米粒及As₂O₃/Fe₃O₄复合纳米粒的制备及表征;②Fe₃O₄磁性纳米粒及As₂O₃/Fe₃O₄复合纳米粒热动力学检测;③Fe₃O₄磁性纳米粒及As₂O₃/Fe₃O₄复合纳米粒生物相容性评价;④As₂O₃/Fe₃O₄复合纳米粒热化疗对培养人宫颈癌Hela细胞治疗作用的研究;⑤As₂O₃/Fe₃O₄复合纳米粒热化疗治疗异种移植性宫颈癌的研究;⑥As₂O₃/Fe₃O₄复合纳米粒热化疗抑制宫颈癌转移相关基因表达的研究。[方法]①采用化学共沉淀法制备Fe₃O₄磁性纳米粒,采用浸渍法制备As₂O₃/Fe₃O₄复合纳米粒,运用透射电子显微镜（transmission electron microscope,TEM）、X射线衍射分析（x-ray diffraction,XRD）、扫描电子显微镜（scanning electron microscopy,SEM）、能谱仪（energy dispersive spectrometer, EDS）及图象分析等技术进行特性研究;②在交变磁场（alternating magnetic field,AMF）作用下观察不同浓度的Fe₃O₄及As₂O₃/Fe₃O₄纳米磁流体的体外升温情况;③采用MTT实验、溶血试验、微核试验、小鼠LD50 （median lethal dose）检测等方法评价Fe₃O₄磁性纳米粒及As₂O₃/Fe₃O₄复合纳米粒的生物相容性;④通过倒置显微镜、电子显微镜、MTT （methyl thiazolyl tetrazolium）实验、流式细胞仪（flow cytometry,FCM）检测,评价As₂O₃/Fe₃O₄复合纳米粒热化疗对体外培养人宫颈癌Hela细胞的治疗作用;⑤将As₂O₃/Fe₃O₄复合纳米粒注入到裸鼠人宫颈癌移植瘤中,经AMF作用3次,6w后测肿瘤质量抑制率及体积抑制率,通过HE切片组织学检查观察治疗后肿瘤组织的形态改变。⑥采用RT-PCR检测人宫颈癌Hela细胞经As₂O₃/Fe₃O₄复合纳米粒热化疗后CD44v6、VEGF-C、及MMP-9的表达情况,初步探讨As₂O₃/ Fe₃O₄复合纳米粒热化疗的抗肿瘤转移机制。[结果]①制备的Fe₃O₄磁性纳米粒表征:TEM检测显示Fe₃O₄磁性纳米粒为圆形、电子密度高、大小较一致、分散良好;图像分析仪粒径统计学分析其平均粒径约为20nm;EDS证实制备的Fe₃O₄磁性纳米粒只含有Fe与O两种元素;XRD显示制备的纳米粒子各衍射峰所对应的面间距（d值）与粉末衍射标准联合会（JCPDS）编制的代表物质为尖晶石型磁性Fe₃O₄的PDF（Powder Diffraction File）卡片d值吻合一致。②制备的As₂O₃/Fe₃O₄复合纳米粒表征:TEM检测显示为圆形、分散良好;图像分析仪粒径统计学分析其平均粒径约为50nm;EDS证实制备的纳米材料含有As、Fe及O三种元素。③体外升温试验:在输出电流20A,输出频率230kHz的AMF作用下不同浓度的Fe₃O₄磁性纳米粒及As₂O₃/ Fe₃O₄复合纳米粒混悬液能升温到42℃～65℃,且加热40min后温度能恒定在某一水平不发生改变。④L-929细胞MTT实验、溶血试验、微核实验证实Fe₃O₄磁性纳米粒无溶血作用、毒性低及无致畸致突变作用、符合生物材料的安全性要求,As₂O₃/Fe₃O₄复合纳米粒亦无溶血作用、无致畸致突变作用;小鼠LD50检测发现Fe₃O₄磁性纳米粒LD50为7.57g/kg,As₂O₃/Fe₃O₄复合纳米粒LD50为更多还原

【Abstract】 [Objective]①To study the preparation and characterization of Fe₃O₄ magnetic nanoparticles and nanosized As₂O₃/Fe₃O₄ complex.②Thermodynamic test of various doses of Fe₃O₄ magnetic nanoparticles and nanosized As₂O₃/Fe₃O₄ complex.③To evaluate the biocompatibility of Fe₃O₄ magnetic nanoparticles and nanosized As₂O₃/Fe₃O₄ complex.④To investigate the therapeutic effects of thermochemotherapy of nanosized As₂O₃/Fe₃O₄ complex on the cultured cervical cancer Hela cells.⑤To investigate the therapeutic effects on xenograft cervical cancer treated by thermochemotherapy of nanosized As₂O₃/Fe₃O₄ complex.⑥To study the inhibition of the gene expression related cancerometastasis treated by thermochemotherapy of nanosized As₂O₃/Fe₃O₄ complex.[Methods]①Fe₃O₄ magnetic nanoparticles were prepared by the technique of chemical co-precipitation and nanosized As₂O₃/Fe₃O₄ complex was prepared by the impregnation process, their characteristics were observed by means of transmission electron microscope（TEM）, x-ray diffraction（XRD）, scanning electron microscopy（SEM）, energy dispersive spectrometer（EDS） and computer color magic image analysis system.②Thermodynamic test was used to observe temperature change of various doses of Fe₃O₄ nanoparticles and nanosized As₂O₃/Fe₃O₄ complex under alternating magnetic field （AMF）.③MTT assay, haemolysis test, micronucleus test, and LD50 calculation of mice were used to evaluate the biocompatibility of Fe₃O₄ nanoparticles and nanosized As₂O₃/Fe₃O₄ complex.④The therapeutic effects on cultured Hela cells treated by thermochemotherapy of nanosized As₂O₃/Fe₃O₄ complex were evaluated by inverted microscope, electron microscope, MTT test and flow cytometry.⑤After injected into xenograft human cervical cancer of nude mice respectively, nanosized As₂O₃/Fe₃O₄ complex was exposed to alternating magnetic field for three times. 6 weeks later, the inhibition rate of the mass and volume of the tumors was calculated. At the same time, histological changes of tumors of tumor-bearing nude mice were observed under light microscope.⑥To study the potential mechanism of anti-cancerometastasis of thermochemotherapy of nanosized As₂O₃/Fe₃O₄ complex, the expression of CD44v6, VEGF-C ,and MMP-9 were detected by RT-PCR technology .[Results]①The self-prepared Fe₃O₄ nanoparticles are approximately spherical, high electron-dense, uniform in size and have good dispersibility observed TEM, which average diameter is about 20nm更多还原