虫草素和阿霉素诱导EBV裂解感染的作用机制及应用研究

发布时间：2018-02-21 00:20

本文关键词： EBV 病毒裂解感染诱导疗法虫草素阿霉素 C/EBPβ　出处：《中国农业大学》2017年博士论文　论文类型：学位论文

【摘要】：EB病毒(Epstein-Barrvirus,EBV)是1964发现的一种人类γ疱疹病毒,EBV在全球人群中的感染率高达95%以上,并以潜伏感染状态在宿主体内终身携带。EBV感染与多种人类疾病相关,而且EBV是第一种被发现的肿瘤相关病毒。全球每年20万例新发肿瘤与EBV相关,并且1.8%左右的癌症死亡是由EBV相关肿瘤所导致。免疫抑制和免疫缺陷人群更易发生EBV阳性肿瘤;与发达地区相比,发展中地区具有更高的EBV相关肿瘤发生率。我国是EBV阳性肿瘤高发国度,尤其是鼻咽癌和胃癌,这对人民健康造成巨大威胁。鉴于EBV阳性肿瘤细胞内多含有处于潜伏感染状态的EBV,而且EBV同时是一种溶瘤病毒,这便提供了通过诱导EBV进入裂解感染状态而杀伤肿瘤细胞的治疗方式。该治疗方式已被多项临床实验证实行之有效,可以明显改善患者的生存质量并延长生存时间。然而目前临床上可供选用的诱导EBV裂解感染的药物还十分有限,因此亟需开发新的药物用于治疗EBV阳性肿瘤。虫草素(cordycepin)是从传统中药冬虫夏草中提取的单体小分子化合物,具有抗炎、抗肿瘤和免疫调节等多种生物学作用。本文通过研究发现,虫草素可以通过PKC、p38 MAPK以及MEK等信号通路激活肿瘤细胞内的EBV进入裂解感染期,并且虫草素对EBV阳性肿瘤细胞具有一定的选择杀伤作用。联合作用实验显示,虫草素可以明显增强传统化疗药物阿霉素(doxorubicin)激活EBV裂解感染的能力,同时虫草素可以显著增强阿霉素对EBV阳性肿瘤细胞的杀伤能力。通过分子机制研究发现,虫草素和阿霉素联合使用可以激活细胞内的PKC-p38 MAPK信号通路,并介导EBV转录激活因子BZLF-1的转录表达。进一步研究发现虫草素和阿霉素在磷酸化激活p38 MAPK以及下游转录因子C/EBPβ的过程中具有联合增强作用。结合以往实验和序列分析,发现在BZLF-1的启动子Zp上具有多个C/EBPβ结合位点;通过荧光素酶报告载体实验和siRNA敲低实验研究发现,转录因子C/EBPβ介导BZLF-1转录以及EBV裂解感染激活过程。最后,动物实验表明虫草素和阿霉素联合使用对EBV阳性肿瘤具有明显的治疗作用,不仅可以明显抑制小鼠体内EBV阳性肿瘤的形成和生长,而且虫草素可以明显减缓阿霉素所造成的小鼠体重下降问题。以上研究结果表明,天然小分子化合物虫草素可以诱导EBV裂解感染,而且虫草素和传统化疗药物阿霉素联合使用对于激活EBV裂解感染具有明显的联合增强作用,体内实验证实虫草素和阿霉素联合使用对于EBV阳性肿瘤具有明显的治疗效果,本文同时阐明了虫草素与阿霉素联合使用通过PKC-p38 MAPK-C/EBPβ信号通路激活EBV裂解感染的分子机制。以上研究结果为EBV阳性肿瘤的治疗提供了重要实验依据和临床应用参考。
[Abstract]:Epstein-Barr virus (EBV) is a human gamma-herpesvirus (EBV) found in 1964. The infection rate of EBV in the global population is as high as 95%. Epstein-Barrvirus.EBV infection in the host is associated with many human diseases. EBV is the first tumor-associated virus found in the world. 200,000 new tumors are associated with EBV every year, and about 1.8% of cancer deaths are caused by EBV related tumors. Immunosuppressive and immunodeficient people are more likely to develop EBV positive tumors. Compared with the developed regions, the developing regions have a higher incidence of EBV related tumors. China is a country with high incidence of EBV positive tumors, especially nasopharyngeal carcinoma and gastric cancer. This poses a great threat to people's health. Given that EBV positive tumor cells often contain potentially infected EBVs, and EBV is also a tumor lysate virus, This provides a way to kill tumor cells by inducing EBV to enter the state of lytic infection, which has been proven to be effective in many clinical trials. It can significantly improve the quality of life and prolong the life span of patients. However, there are very few drugs available in clinic to induce EBV lytic infection. Therefore, it is urgent to develop new drugs for the treatment of EBV positive tumors. Cordycepin (Cordycepin) is a small monomer compound extracted from traditional Chinese medicine Cordycepin, which has anti-inflammatory properties. In this paper, we found that Cordycepin can activate the EBV in tumor cells to enter the lytic infection stage through signal pathways such as PKCnp38 MAPK and MEK. In addition, Cordycepin has a selective killing effect on EBV positive tumor cells. The combined effect of Cordycepin on EBV positive tumor cells showed that Cordycepin could significantly enhance the ability of traditional chemotherapeutic drug doxorubicin to activate EBV lytic infection. At the same time, Cordycepin could significantly enhance the cytotoxicity of adriamycin to EBV positive tumor cells. It was found that the combination of Cordycepin and Adriamycin could activate the PKC-p38 MAPK signaling pathway in the cells. Furthermore, it was found that Cordycepin and adriamycin could enhance the phosphorylation of p38 MAPK and downstream transcription factor C / EBP 尾, combining with previous experiments and sequence analysis. It was found that there were several C / EBP 尾 binding sites on the promoter Zp of BZLF-1. Through luciferase report vector experiment and siRNA knockdown experiment, it was found that the transcription factor C / EBP 尾 mediated BZLF-1 transcription and the activation of EBV lytic infection. Animal experiments showed that the combination of cordycepin and adriamycin had a significant therapeutic effect on EBV positive tumors, and could not only inhibit the formation and growth of EBV positive tumors in mice. In addition, Cordycepin can significantly reduce the weight loss caused by adriamycin in mice. The above results show that Cordycepin, a natural small molecular compound, can induce EBV cleavage infection. In addition, the combination of Cordycepin and adriamycin in combination with traditional chemotherapeutic drugs can significantly enhance the activation of EBV lytic infection. In vivo, the combination of Cordycepin and Adriamycin has a significant therapeutic effect on EBV positive tumors. The molecular mechanism of the combination of cordycepin and adriamycin to activate EBV lytic infection through PKC-p38 MAPK-C/EBP 尾 signaling pathway was also elucidated. These results provide an important experimental basis and clinical application reference for the treatment of EBV positive tumors.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R730.5

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