【作者】 周菊梅；

【导师】 唐劲天； 廖遇平；

【作者基本信息】 中南大学 ， 肿瘤学， 2009， 博士

【摘要】 研究背景宫颈癌是女性生殖系统发病率最高的恶性肿瘤,与高危型HPV感染直接相关,近年来发病率呈上升和年轻化的趋势。晚期宫颈癌以放化疗为主。由于放化疗抵抗以及放化疗的毒性反应,晚期宫颈癌的5年生存率为50%左右。热疗引起细胞凋亡和坏死,同时具有放化疗增敏优势,在宫颈癌的综合治疗中提高了5年生存率和局部控制率,而远期毒性反应没有增加。热疗对宫颈癌的作用机制尚不清楚。传统的热疗认为43℃是大多数细胞发生凋亡的临界点,但近期研究发现,43℃热疗并不是对所有种类的癌细胞均具有杀伤作用。细胞对热疗的敏感性与细胞的种类和基因特征有关。探索不同细胞株对热疗的敏感性有助于临床个体化的热疗方案。磁热疗是一种新兴的热疗手段,与传统的热疗方式相比具有靶向定位升温的优势,而对周围正常组织的损伤小,在国外的前列腺癌等Ⅱ期临床试验中显示较好的疗效,但是对宫颈癌的研究未见报道。因此,我们采用不同温度体内外加热宫颈癌细胞,探讨不同温度热疗对细胞凋亡和增殖的影响,以及凋亡的内在作用机制,为热疗在宫颈癌的临床运用提供部分理论依据。第一章不同温度热疗对宫颈癌Caski细胞凋亡和增殖的影响目的探讨不同温度热疗对宫颈癌Caski细胞凋亡、增殖以及细胞周期的影响。方法采用水浴对宫颈癌Caski细胞行43℃、45℃、47℃实验组和37℃对照组热疗,四甲基偶氮唑蓝（MTT）检测细胞增殖抑制率,流式细胞仪测定细胞凋亡率、坏死率和细胞周期的变化,免疫组化检测增殖细胞核抗原（PCNA）的表达。结果45℃,47℃组的细胞增殖抑制率在24h、48h、72h、96h分别为76.11±5.32%、81.08±4.03%、74.71±3.78%、72.16±4.75%和88.57±3.77%、91.33±2.43%、91.09±1.17%、92.05±1.67%,明显高于43℃组的26.57±3.46%、25.49±2.76%、22.80±3.16%、26.87±4.01%（P<0.05）。45℃与47℃组的细胞增殖抑制率之间的差异无显著性（P>0.05）。45℃组的细胞凋亡率最高,为12.82±1.77%,与其它各组之间的差异具有显著性（P<0.05）。47℃组的细胞坏死率最高,为39.15±5.67%,各组之间的差异均有显著性（P<0.05）。43℃、45℃、47℃组的G₁期细胞比对照组明显减少（P<0.05）,而S期细胞明显增加（P<0.05）。PCNA的表达随着热疗温度的增加而降低。45℃,47℃组的平均光密度值（AOD）分别是0.228±0.022、0.214±0.018,与对照组和43℃相比差异具有显著性（P<0.05）。结论45℃及以上的热疗能够明显抑制Caski细胞的增殖,增加凋亡率和坏死率,细胞周期阻滞在S期,降低PCNA的表达。第二章热疗诱导Smac/DIABLO表达促进Caski细胞凋亡目的研究宫颈癌Caski细胞经过不同温度热疗后凋亡相关基因p53、Smac/DIABLO、survivin、caspase-3的表达变化。方法采用水浴对宫颈癌Caski细胞行43℃、45℃、47℃热疗40min,实时荧光定量PCR检测不同温度热疗后继续培养2h、6h、12h、24h HPV16E6、p53、Smac/DIABLO、survivin、caspase-3在mRNA水平的表达变化。Western Blotting检测不同温度热疗后2h、6h、12h、24h Smac/DIABLO、survivin、caspase-3在蛋白水平的表达变化。免疫组化检测不同温度热疗后24h caspase-3蛋白在细胞内的表达变化。结果（1） 43℃热疗后各时间点HPV16E6、Smac/DIABLO、survivin、caspase-3 mRNA的表达均比对照组增加（P<0.05）。与对照组相比,43℃热疗后2h出现survivin蛋白的表达降低,其后survivin蛋白的表达反而上调（P<0.05）。43℃热疗后6h和24h Smac/DIABLO蛋白的表达明显增加（P<0.05）;而caspase-3蛋白表达增加出现在热疗后12-24h（P<0.05）。（2） 45℃热疗后各时间点HPV16E6 mRNA、survivin mRNA和蛋白的表达比对照组降低（P<0.05）。45℃热疗后12-24h Smac/DIABLO和caspase-3 mRNA的表达比对照组明显增加（P<0.05）。45℃热疗后6h Smac/DIABLO和caspase-3蛋白表达明显上调,并维持至热疗后24h（P<0.05）。（3） 47℃热疗后各时间点HPV16E6、Smac/DIABLO、survivin mRNA的表达均比对照组明显降低（P<0.05）;而caspase-3 mRNA在热疗后24h比对照组增加（P<0.05）。47℃热疗后2h Smac/DIABLO蛋白的表达和2-6hcaspase-3蛋白的表达比对照组明显增加（P<0.05）,之后两者的表达明显低于对照组（P<0.05）。47℃热疗后在各时间点survivin蛋白的表达显著降低甚至缺失（P<0.05）。（4） 43℃、45℃和47℃热疗后野生型p53 mRNA的表达比对照组明显减少（P<0.05）。（5）43℃和45℃热疗后24h caspase-3的AOD值比37℃和47℃明显增加（P<0.05）。而45℃和47℃热疗后caspase-3的核阳性指数增加,与37℃和43℃相比,差异具有显著性（P<0.05）。结论宫颈癌Caski细胞中Smac/DIABLO、survivn和caspase-3在mRNA和蛋白水平的表达变化与热疗温度和培养时间有关。45℃以上的热疗抑制HPV16E6 mRNA的表达。热疗后Smac/DIABLO通路诱导的Caski细胞凋亡可能不依赖于野生型p53基因的调控。热疗后caspase-3从细胞浆易位至细胞核是细胞凋亡的重要事件。第三章不同温度磁热疗对人宫颈癌裸鼠皮下移植瘤的影响目的探讨不同温度磁热疗对人宫颈癌裸鼠皮下移植瘤的作用。方法用人宫颈癌Hela细胞株建立裸鼠皮下移植瘤模型,将Fe₃O₄磁性纳米颗粒直接注入肿瘤组织内,在交变磁场中进行磁感应加热治疗,分别控温在43℃和47℃持续30min,3天后重复加热一次,比较43℃组磁热疗组（43℃MFH）、47℃磁热疗组（47℃CMFH）、单纯磁流体组（MF组）、生理盐水（NS组）和对照组的肿瘤体积的大小、肿瘤体积抑制率、生存期和PCNA的阳性指数。结果热疗后的第1-4周47℃MFH组的肿瘤体积抑制率分别为47.05%、89.21%、88.55%、87.19%;43℃MFH组的分别为32.26%、52.64%、48.84%、38.42%。与对照组相比,43℃MFH组和47℃MFH组均能抑制肿瘤的生长（P<0.05）。与43℃MFH组比较,47℃MFH组抑瘤作用更显著,有33.33%的肿瘤消退率（P<0.05）。43℃MFH组和47℃MFH组的生存期分别为36.67±1.28d、49.33±3.19d,比对照组的27.33±1.41d明显延长（P<0.05）。而47℃MFH组与43℃MFH组的生存期之间的差异亦具有显著性（P<0.05）。PCNA的阳性指数在47℃MFH组显著降低为12.33±3.40%,与其它组相比,差异具有显著性（P<0.05）。结论43℃MFH组和47℃MFH组均能抑制人宫颈癌裸鼠皮下移植瘤的生长,延长裸鼠的生存期,抑制PCNA的增殖,但是以47℃MFH组的作用更为显著,甚至可以达到肿瘤完全消退的效应。更多还原

【Abstract】 Background: Cervical cancer has become one of the most prevalent gynecological neoplasma and the main cause is persistent infection of high-risk human papillovirus. The incidence of cervical cancer experiences a significant rise and sufferers become younger in average age. The primary possible treatment of advanced cervical cancer may be a combination of radiation and chemotherapeutic agent such as cisplatin. However, owing to lack of acknowledgment of tumor resistance and side effects of chemo-radiotherapy, the 5-year overall survival rate is only 50% for advanced cervical cancer. When tumors cells expose to higher thermal conditions, more apoptosis and necrosis are often induced and cells usually exhibit hypersensitivity to radiation and/or chemotherapy after heat treatment. The combined treatment of advanced cervical cancer has resulted in long-term major improvement in local control and survival without increasing late toxicity.The mechanism of hyperthermia on cervical cancer is not clear. Traditional hyperthermia believes that there is an apoaptosis transition at 43℃for majority of cells. However, recent studies found that hyperthermia at 43℃could not kill all kinds of cancer cells. The cell sensitivity to hyperthermia is correlated with category of cell and feature of gene. Investigation for cell sensitivity to hyperthermia contributes to the clinic treatment.A new nanotechnology based thermotherapy using magnetic nanoparticles, also referred to as magnetic fluid hyperthermia （MFH） was developed. Compared with traditional hyperthermia, magnetic fluid hyperthermia is a better temperature homogeneity and targeted orient in the tumor with less damnification to peripheral normal tissues. It shows significant effects in phaseⅡclinical trial, but there are no reports about the investigation of cervical cancer.Therefore, in order to find the appropriate temperature for treatment cervical cancer, we heat cervical cancer cells at different temperature in vitro and in vivo then explore the effect on apoptosis and proliferation of cells. Objective To investigate the effect of hyperthermia on the apoptosis, proliferation and cell cycle of Caski cells.Methods Caski cells were heated at 43℃, 45℃, 47℃and 37℃in temperature-controlled water bath, MTT assay, flow cytometry and immunohistochemical technique were applied to analyze the growth inhibition, apoptosis rate,necrosis rate, cell cycle as well as expression of PCNA.Results The inhibitive rates of cell growth after 24h, 48h, 72h, 96h at 45℃and 47℃was 76.11±5.32%, 81.08±4.03%, 74.71±3.78%, 72.16±4.75% and 88.57±3.77%, 91.33±2.43%, 91.09±1.17%, 92.05±1.67% respectively, which was remarkably higher than that of 26.57±3.46%, 25.49±2.76%, 22.80±3.16%, 26.87±4.01% at 43℃（P<0.05）. There is no significant difference in inhibitive effect of cell growth between 45℃and 47℃（P<0.05）. While the highest apoptosis rate happened at 45℃, the highest necrosis rate was observed at 47℃, both with statistical significance as compared with other groups （P<0.05）. Results from flow cytometry indicate G₁ phase cells after 24h at 43℃, 45℃and 47℃were remarkably lower than that of the control group （P<0.05）. However, S phase cells was remarkably higher as compared with the control group （P<0.05）. Immunohistochemical analysis demonstrates that the expression of PCNA decreased with the increase of the temperature for heating treatment. When compared with the controlled group and 43℃group, the expression was descented at 45℃and 47℃, which was respectively 0.228±0.022 and 0.214±0.018（P<0.05）.Conclusions The hyperthermia at 45℃or higher termperaturecould remarkably inhibit the proliferation of Caski cells , increase the apoptosis rates and necrosis rates, arrest the cells cycle in S phase and decrease the expression of PCNA. Objective To study the effect of temperatures for in vitro heating treatment of Caski cells on the protein and mRNA expression of p53, Smac/DIABLO, survivin and caspase-3.Methods Caski cells were harvested at 2h, 6h, 12h and 24h after heating treatment at 43℃, 45℃, 47℃and 37℃for 40min in temperature-controlled water bath, Real time PCR, western blotting, as well as immunohistochemical technique were employed to determine the mRNA and protein expressions of HPV16E6, p53, Smac/DIABLO, survivin and caspase-3.Results （1） The mRNA expression of HPV16E6, Smac/DIABLO, survivin and caspase-3 was increased within 24h at 43℃when compared with the control group （P<0.05）. However, protein expression demonstrated time-dependent under the 43℃heating treatment. Expression of survivin was increased from 6h to 24h with statistical significance （P<0.05）, though a decreased expression was observed at 2h. Expression of Smac/DIABLO at 6h and 24h was higher than that of the control group （P<0.05）. When compared with the control group, caspase-3 was increased from 12h to 24h （P<0.05）. （2） At 45℃, the heating treatment could inhibit the mRNA expression of HPV16E6 and survivin, as well as protein expression of survivin within 24h with statistical significance （P<0.05）. When compared with the control group, the mRNA expression Smac/DIABLO and caspase-3 was increased 12-24h after heating treatment at 45℃（P<0.05）. Meanwhile, the protein expression of Smac/DIABLO and caspase-3 was increased from 6h until 24h after heating （P<0.05）. （3） When compared with the control group, the mRNA expression of HPV16E6, Smac/DIABLO and survivin was remarkably decreased within 24h at 47℃（P<0.05）, but the mRNA expression of caspase-3 was increased at 24h （P<0.05）. Protein expression of Smac/DIABLO at 2h and caspase-3 from 2h to 6h was increased with statistical significance after heating treatment （P<0.05）. Protein expression of survivin was remarkably inhibited （P<0.05）. （4） When compared with the control group, a reductive mRNA expression of wild type p53 was observed regardless of the heating temperature （P<0.05）. （5） Compared with the control group and the 47℃group, AOD value of caspase-3 expression for the 43℃and 45℃groups was increased （P<0.05）. Nucleus positive index of caspase-3 expression for the 45℃and 47℃groups was higher than that of the control group and the 43℃group （P<0.05）.Conclusions Protein and mRNA expression of Smac/DIABLO, survivin and caspase-3 was dependent on the temperature and culture time. The hyperthermia at 45℃or higher temperature could inhibit the mRNA expression of HPV16E6. However, no noticeable change was observed on the mRNA expression of wild-type p53 subjected to heating treatment regardless of the temperature, which indicating the apoptosis induced by Smac/DIABLO after heating treatment could not depend on wild-type p53 gene. Caspase-3 translocated from cytoplasm to the nucleus was an important event for the apoptosis of the cells.Objective To study the temperature effect of magnetic fluid hyperthermia （MFH） at 43℃or 47℃in a murine xenograft model of human cervical cancer.Methods Murine xenograft model of human cervical cancer was established by transplanting minced fragment of tumours into the subcutaneous tissue of the thigh of nude mouse using a trocar. The tumor-bearing mice then underwent radiation by an alternative magnetic filed （AMF） after the Fe₃O₄ magnetic fluid （MF） was locally injected in the tumor area. The parameters of the AMF were carefully adjusted until a local tumor temperature （43℃or 47℃） was maintained for 30 min. The MFH was performed twice with 72h interval. The tumour volume, rate of tumor volume inhibition, mice survival and the index of PCNA were examined.Results MFH at 43℃or 47℃could inhibit the tumor growth. Compared with 43℃MFH, 47℃MFH had a greater inhibitive effect on tumor growth （P<0.05）. The inhibitive rates of tumor volume from the first week to the fourth week at 43℃MFH group and 47℃MFH grou was 32.26%, 52.64%, 48.84%, 38.42% and 47.05%, 89.21%, 88.55%, 87.19% respectively. The survival of 47℃MFH group and 43℃MFH group were more prolonged than that of control group （P<0.05）. When compared with the other groups, the expression of PCNA was remarkably decreased at 47℃MFH group （P<0.05）.Conclusion MFH at 43℃or 47℃could inhibit the tumor growth, prolong survival as well as decreased the expression of PCNA with more remarkable effect at 47℃MFH group, in which the total tumour regression was observed.更多还原