氧化亚铜纳米粒对宫颈癌的治疗及其机制研究

发布时间：2018-04-26 22:19

本文选题：宫颈癌 + 治疗　；参考：《第二军医大学》2017年硕士论文

【摘要】：宫颈癌,是女性最常见的恶性肿瘤之一,全球每年有近50万的女性被诊断为宫颈癌,在我国,其发病率和死亡率占全球近1/4,严重威胁了我国女性的健康。目前认为,宫颈癌的发病与人乳头瘤病毒(HPV,Human papillomavirus)高危型密切相关。已有统计数据表明,HPV16,18,31,33,35,39,45,51,52,56,58,59,68,73和82型等均与宫颈癌有关,而其中HPV16型、18型分别在宫颈癌中感染率约为50%和20%;目前大量研究数据表明,HPV高危型中的E6及E7蛋白在宫颈癌的恶性维持中起着至关重要的作用,其中,高危型HPV E6可经泛素化途径诱导肿瘤抑制因子p53的降解,以消除其生长抑制信号。E7则可磷酸化Rb蛋白,从而发挥促进肿瘤增殖等作用。目前宫颈癌的治疗方法主要有手术及放化疗。手术治疗主要适用于宫颈癌早期病人。放疗虽然适用于各期病人,但是由于放疗可对阴道及卵巢造成不可逆的损伤,因此,也限制了放疗在年轻有生育要求的宫颈癌患者的应用。鉴于宫颈癌对化疗仅中度敏感,所以,目前化疗主要作为宫颈癌晚期患者的辅助性及姑息性治疗手段。因此,寻找治疗宫颈癌的新途径及新方法具有显著的意义。众所周知,纳米,作为一个长度计量单位,仅为十亿分之一米。当物质进入纳米尺度,则会出现明显的性能方面的改变。而纳米医学,则是把纳米科学技术与现代医学的研究融合在一起新兴学科。在纳米治疗的领域当中,纳米药物表现出传统化疗药物所不具备的优点,如粒径小、渗透能力强等,抗肿瘤纳米药物既可直接作为抗肿瘤药物作用于肿瘤组织,也可经改造为载药体系,在细胞水平和动物水平均表现出良好的肿瘤治疗应用前景。目前大量的研究证实,纳米粒可通过干扰参与细胞增殖、凋亡、转录因子等相关蛋白的表达,从而作用于特定的信号转导通路,发挥出特异的生物学活性。金纳米粒(Au NPs)可抑制MAPK信号通路从而抑制肿瘤的增殖;纳米氧化锌(Zn O)可通过抑制Akt、Erk1/2的磷酸化,激活JNK、P38等,抑制肿瘤的转移。大量的研究表明纳米药物可作用于多条信号转导通路,具有选择性的药理活性,因此,纳米抗肿瘤药物具有深入探索和研究的巨大潜力和价值。氧化亚铜纳米粒(Cuprous Oxide Nanoparticles,CONPs)是一种含铜纳米药物,分子式为Cu2O,目前已有研究表明,氧化亚铜纳米粒CONPs可抑制恶性肿瘤细胞的增殖和转移,并且研究证实氧化亚铜纳米粒CONPs可通过线粒体介导的细胞凋亡信号通路发挥抗肿瘤的作用。本课题通过cck8实验,transwell实验,细胞凋亡、细胞周期检测,发现氧化亚铜纳米粒CONPs可显著抑制宫颈癌细胞的增殖,迁移和侵袭,并且可诱导宫颈癌细胞凋亡,阻滞宫颈癌细胞周期。通过裸鼠荷瘤实验发现,氧化亚铜纳米粒CONPs不仅可抑制裸鼠肿瘤的增殖,且与顺铂治疗组相比,氧化亚铜纳米粒CONPs治疗组无明显体重下降等副作用。体内外结果实验都提示着氧化亚铜纳米粒CONPs在宫颈癌治疗中有着良好的应用前景。为了更好的研究氧化亚铜纳米粒CONPs治疗宫颈癌的作用机制,为临床转化应用打下坚实的基础,我们通过透射电镜(TEM)分析发现,氧化亚铜纳米粒CONPs可穿过细胞膜,聚集在细胞的线粒体,内质网等部位。并且发现,随着氧化亚铜纳米粒CONPs浓度的增大,细胞自噬体的产生也随之增多。通过激光共聚焦及流式分析发现,氧化亚铜纳米粒CONPs可损伤线粒体导致线粒体膜电位下降,并且通过自噬流研究也进一步确认了随着氧化亚铜纳米粒CONPs浓度的增大,细胞内自噬体的产生是随之增多的。通过Western Blot检测LC3等自噬相关蛋白,结果也与之前相符,即随着氧化亚铜纳米粒CONPs处理浓度的增大,LC3 II/I的比例也随之增加,并且,随着氧化亚铜纳米粒CONPs处理时间的延长,LC3 II/I的比例也有着一定程度的增加。鉴于目前自噬的发生与AKT/m TOR通路有着密切的关系,我们检测了相关蛋白的表达,实验结果分析发现,CONPs可通过抑制AKT/m TOR通路,降低AKT、m TOR的磷酸化水平,从而发挥抗肿瘤的作用。综上所述,本课题研究表明CONPs可抑制宫颈癌的增殖,诱导宫颈癌细胞的凋亡,并且可通过抑制AKT/m TOR通路发挥作用。
[Abstract]:Cervical cancer is one of the most common malignant tumors in women. Nearly 500 thousand of women in the world are diagnosed with cervical cancer in the world every year. In China, the incidence and mortality of the women are nearly 1/4 worldwide, which seriously threaten the health of women in our country. At present, the incidence of cervical cancer is closely related to the high-risk type of human papillomavirus (HPV, Human papillomavirus). Statistics show that HPV16,18,31,33,35,39,45,51,52,56,58,59,68,73 and type 82 are all related to cervical cancer, and the infection rate of type HPV16 and type 18 in cervical cancer is about 50% and 20% respectively. A large number of research data show that the E6 and E7 protein in the high risk type of HPV plays a vital role in the maintenance of cervical cancer, of which the high risk is at high risk. Type HPV E6 can induce the degradation of tumor suppressor factor p53 through ubiquitination, so as to eliminate its growth inhibition signal.E7, which can phosphorylate Rb protein, and thus play a role in promoting tumor proliferation. At present, the main treatment methods of cervical cancer are operation and radiotherapy and chemotherapy. The operation is mainly applied to early patients with cervical cancer. Although radiotherapy is applicable to all stages of cervical cancer, the treatment is suitable for all stages. Patients, but because radiotherapy can cause irreversible damage to the vagina and ovary, and therefore, it also restricts the use of radiotherapy in young patients with reproductive requirements for cervical cancer. In view of the moderate sensitivity of cervical cancer to chemotherapy, chemotherapy is currently used as an auxiliary and palliative treatment for advanced cervical cancer patients. New approaches and methods for cervical cancer are significant. As we all know, nanoscale, as a measurement unit of length, is only 1/1000000000 meters. When material enters nanoscale, there will be obvious changes in performance. Nanomedicine is a new subject that combines nanotechnology and modern medicine. In the field of nanotherapy, nano drugs show the advantages that traditional chemotherapeutic drugs do not possess, such as small size and strong osmosis. Antitumor nanodrugs can be used as antitumor drugs directly to tumor tissue, and can also be transformed into drug loading systems, and good tumor treatment applications are shown at both the cell level and the animal level. A large number of studies have confirmed that nanoparticles can interfere with the expression of cell proliferation, apoptosis, transcription factors and other related proteins by interfering with specific signal transduction pathways, and play a specific biological activity. Au NPs can inhibit MAPK signaling and inhibit the proliferation of tumor; nano Zinc Oxide (Zn O) can be used. Excessive inhibition of Akt, Erk1/2 phosphorylation, activation of JNK, P38, and so on, inhibit tumor metastasis. A large number of studies have shown that nanometers can play a role in multiple signal transduction pathways and have selective pharmacological activity. Therefore, nano antitumor drugs have great potential and value for exploration and research. Cuprous Oxide Nanoparticl (Cuprous Oxide Nanoparticl) Es, CONPs) is a copper containing nano drug and molecular formula is Cu2O. Currently, it has been shown that cuprous nanoparticles CONPs can inhibit the proliferation and metastasis of malignant tumor cells, and the study confirms that the cuprous nanoparticles CONPs can play an antitumor effect through mitochondrial mediated apoptosis signal. This subject is conducted through CCK8 experiment, tr Answell experiments, apoptosis and cell cycle detection showed that CONPs nanoparticles could significantly inhibit the proliferation, migration and invasion of cervical cancer cells, and induce cervical cancer cell apoptosis and block cervical cancer cell cycle. It was found that oxidized cuprous nanoparticles CONPs can not only inhibit the proliferation of nude mice, but also inhibit the proliferation of tumor in nude mice. Compared with cisplatin treatment group, there is no obvious side effect in the treatment group of cuprous oxide nanoparticles CONPs. Both in vivo and in vivo results suggest that cuprous nanoparticles CONPs has a good prospect in the treatment of cervical cancer. In order to better study the mechanism of the effect of cuprous nanoparticles CONPs in the treatment of cervical cancer, it can be used for clinical transformation. On a solid basis, we found through transmission electron microscopy (TEM) analysis that the cuprous nanoparticles CONPs can pass through the cell membrane and gather in the mitochondria, endoplasmic reticulum and other parts of the cell. And it is found that the production of the cell autophagic increases with the increase of the concentration of cuprous oxide nanoparticles CONPs. The cuprous nanoparticles CONPs can damage mitochondria and lead to the decrease of mitochondrial membrane potential and further confirm that the production of autophagic in the cell is increased with the increase of CONPs concentration of cuprous oxide nanoparticles. The detection of autophagic related proteins such as LC3 by Western Blot is also consistent with the results before. With the increase of CONPs concentration of cuprous nanoparticles, the proportion of LC3 II/I increased, and the proportion of LC3 II/I increased with the prolongation of the CONPs treatment time of cuprous nanoparticles. In view of the close relationship between the occurrence of autophagy and the AKT/m TOR pathway, we detected the expression of the related proteins. The experimental results show that CONPs can reduce the phosphorylation level of AKT, m TOR by inhibiting the AKT/m TOR pathway, and thus exerts anti-tumor effect. In summary, this study shows that CONPs can inhibit the proliferation of cervical cancer and induce apoptosis of cervical cancer cells, and can play a role in the inhibition of AKT/m TOR pathway.

【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.33

【参考文献】