【作者】 倪海燕；

【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①Mn_xZn_1-xFe₂O₄磁性纳米粒的制备及表征;②As₂O₃/Mn_0.5Zn_0.5Fe₂O₄复合纳米粒的制备及其特性检测;③As₂O₃/Mn_0.5Zn_0.5Fe₂O₄复合纳米粒联合磁流体热疗对人宫颈癌Hela细胞作用的研究;④As₂O₃/Mn_0.5Zn_0.5Fe₂O₄复合纳米粒联合磁流体热疗对异体人宫颈癌移植瘤作用的研究。方法①采用改良的化学共沉淀法制备Mn_xZn_1-xFe₂O₄磁性纳米粒,透射电子显微镜（transmission electron microscope,TEM）、能谱仪（energy dispersive spectrometer, EDS）及X射线衍射（x-ray diffraction,XRD）、热分析系统进行表征,并在高频交变磁场（AMF）下检测Mn_0.5Zn_0.5Fe₂O₄磁流体热效应情况;②采用浸渍法制备As₂O₃/Mn_0.5Zn_0.5Fe₂O₄复合纳米粒,透射电镜、能谱仪、原子荧光分光光度计、原子吸收分光光度计等分析其特性;③分别将RPMI1640培养液、As₂O₃液、Mn_0.5Zn_0.5Fe₂O₄纳米粒、As₂O₃/Mn_0.5Zn_0.5Fe₂O₄复合纳米粒在AMF下作用于宫颈癌HeLa细胞,MTT、流式细胞分析仪（FCM）检测并比较各组治疗效果;④采用宫颈癌瘤组织块建立移植瘤模型,将上述各组作用于移植瘤,Mn_0.5Zn_0.5Fe₂O₄纳米粒联合MFH组、As₂O₃/ Mn_0.5Zn_0.5Fe₂O₄复合纳米粒联合MFH组的荷瘤鼠在交变磁场下作用3次,每次间隔24小时,7w后颈椎脱臼处死裸鼠,剥离肿瘤,测并计算肿瘤质量抑制率及体积抑制率,同时通过HE切片组织学检查观察肿瘤组织、肿瘤周围组织及裸鼠各主要脏器的形态改变。⑤采用逆转录酶-聚合酶链式反应（RT-PCR）法检测HPV18E7与As₂O₃/Mn_0.5Zn_0.5Fe₂O₄复合纳米粒联合磁流体热疗对宫颈癌作用的关系。结果①制备的Mn_xZn_1-xFe₂O₄磁性纳米粒子呈棕褐色,平均直径30～40nm、近似球形,有良好的磁感应性;XRD显示制备的纳米粒的各X射线衍射谱图的特异峰线的高度和位置与JCPDS（粉末衍射标准联合会）数据库卡片中的Mn_0.5Zn_0.5Fe₂O₄磁性纳米粒子结果一致;EDS证实制备的纳米材料含有Mn、Zn、Fe与O四种元素,无其它杂质;且其在高频交变磁场作用下具有明显的热效应,升温可至肿瘤热疗的有效温度范围（41℃～46℃）,并且可以恒温;②As₂O₃/Mn_0.5Zn_0.5Fe₂O₄复合纳米粒近似球形,粒径在100nm左右。能谱仪证实其包含由锰、锌、铁、氧及砷,无其它成分,其含砷量为0.61%。③体外细胞实验:倒置显微镜观察发现As₂O₃/ Mn_0.5Zn_0.5Fe₂O₄复合纳米粒联合MFH能引起Hela细胞坏死及凋亡,MTT检测显示其能显著抑制HeLa细胞的增殖,流式细胞分析（FCM）检测显示其能诱导人宫颈癌HeLa细胞的凋亡,且其效果较单纯As₂O₃液及单纯MFH明显。而RPMI1640培养液及磁性纳米粒对细胞没有影响。④体内抑瘤实验:As₂O₃/ Mn_0.5Zn_0.5Fe₂O₄复合纳米粒联合MFH组肿瘤体积和质量抑制率分别为92.77%、90.01%,与对照组（CG）、磁性纳米粒组、单纯As₂O₃组和As₂O₃/ Mn_0.5Zn_0.5Fe₂O₄复合纳米粒组相比有明显差异（P<0.01）,且磁性纳米粒联合MFH组和As₂O₃/ Mn_0.5Zn_0.5Fe₂O₄复合纳米粒联合MFH组相比也有明显的差异（P<0.05）。两热疗组的肿瘤组织有明显坏死,而瘤旁及内脏组织均未见明显损伤。⑤RT-PCR显示:As₂O₃终浓度2μM的游离As₂O₃及As₂O₃/ Mn_0.5Zn_0.5Fe₂O₄复合纳米粒组,在循环参数30个条件下,HPV18-E7mRNA的表达明显受抑制,而单纯纳米Mn_0.5Zn_0.5Fe₂O₄处理组,联合或未联合MFH的情况下均不影响HPV18-E7mRNA的表达。结论①成功制备了Mn_0.5Zn_0.5Fe₂O₄磁性纳米粒及As₂O₃/Mn_0.5Zn_0.5Fe₂O₄复合纳米粒;②As₂O₃/Mn_0.5Zn_0.5Fe₂O₄复合纳米粒在高频交变磁场作用下可升温控温治疗宫颈癌;③As₂O₃/Mn_0.5Zn_0.5Fe₂O₄复合纳米粒可同时发挥As₂O₃的细胞毒性作用和磁感应加热的联合定向肿瘤热化疗的治疗作用,效果优于单一治疗,此为临床治疗宫颈癌提供新的方法。更多还原

【Abstract】 Objective①To study preparation and characterization of Mn-Zn ferrite magnetic nanoparticles②To study preparation and characterization of nanosized As₂O₃/Mn0.5Zn0.5Fe2O4 complex③To evaluate the therapeutic effect of As₂O₃/Mn0.5Zn0.5Fe2O4 complex combined with magnetic fluid hyperthermia （MFH） on the human cervival cancer cells④To investigate the therapeutic effect of As₂O₃/Mn0.5Zn0.5Fe2O4 complex combined with MFH on xenograft cervix cancer in nude mice.Methords①With the sulfate as the materials and NaOH as precipitator, Mn0.5Zn0.5Fe2O4 nanoparticles were prepared,which are characterizated by X-ray diffraction（XRD）、transmission electron microscope（TEM） and heat analysis system .Besides, the heat effect was studied in alternating magnetic field in vitro②As₂O₃/Mn0.5Zn0.5Fe2O4 complex was prepared by impregnation process, and its properties were exmined through TEM、EDS and atom fluorescence spectrophotometer, drug releasing test in vitro③MTT assay was performed to examine the effect of nanosized As₂O₃/Mn0.5Zn0.5Fe2O4 complex on the proliferation of Hela cells. By means of FCM, the ratio of apoptotic cells was detected.④After injected into xenograft human cervival cancer on nude mice respectively,Mn0.5Zn0.5Fe2O4 nanoparticles and As₂O₃/Mn0.5Zn0.5Fe2O4 complex were explored to AMF （SP-04C, 200kHz, 4kW, Shenzhen, China） for 30 min. The treatment was given for 3 times at 24h intervals. After 6 weeks, all mice were killed. the inhibition rate of the weight 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.⑤The levels of mRNA of HPVE7 was measured by RT-PCR.Results①the prepared Mn0.5Zn0.5Fe2O4 nanoparticles are spinel Mn-Zn ferrite ,global, average diameter 30⁴0nm. The Mn0.5Zn0.5Fe2O4 nanoparticles have very distinct heat effect in a alternating magnetic field and can rise to a steady temperature for tumor hyperthermia （41℃～46℃）.②The prepared As₂O₃/Mn0.5Zn0.5Fe2O4 complex are about global, average diameter 100nm; As₂O₃content is 0.61%.③Magnetic nano-microspheres containing As₂O₃ could significantly inhibit the proliferation of human Hela cells and promoted its apoptosis in vitro. The cell survival ratio in the group of nanosized As₂O₃/ Mn0.5Zn0.5Fe2O4 （41.69%） was distinctly lower than that of the As₂O₃solution （79.78% p<0.05） and that of the Mn0.5Zn0.5Fe2O4 nanoparticle magnetic fluid （59.48% p<0.05）. The apoptosis ratio of magnetic nano-microspheres containing As₂O₃ （31.26%） was obviously higher than that of the other two groups （7.83%, 14.51%）. The single magnetic material had no effect on cell growth in vitro④Animal experiment showed that the mass inhibitory ratio of nanosized As₂O₃/Mn0.5Zn0.5Fe2O4 was 96.70%, while As₂O₃ group and MFH group were 67.03%, 79.99% respectively. By means of SPSS11.5 software, the results showed that each group had distinct difference （p<0.01）. On all accounts, the nanosized As₂O₃/Mn0.5Zn0.5Fe2O4 has both cytoxicity effects and the heating effects resulted from the radiofrequency-induced hyperthermia. And it has better effects on cervix cancer than either using As₂O₃ or hyperthermia alone.⑤The results of RT-PCR implies that 2μM As₂O₃ can inhibite the expression of HPV18-E7 oncogene, but this effect was not reinforced when combined with hyperthermia.Conclusions①Mn0.5Zn0.5Fe2O4nanoparticle and nanosized As₂O₃/Mn0.5Zn0.5Fe2O4 complex could be prepared by improved co-precipitation process and impregnation process respectively.②Taking advantage of更多还原