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海洋细菌胞外多糖EPS11抗肿瘤转移机制研究
王菊
Subtype硕士
Thesis Advisor孙超岷
2019-05-14
Degree Grantor中国科学院大学
Place of Conferral中国科学院海洋研究所
Degree Name理学硕士
Degree Discipline海洋生物学
Keyword海洋细菌 多糖eps11 癌症 黏附 转移
Abstract

    癌症作为世界范围内的主要公共卫生问题之一,严重威胁人类生命健康,并且其发病率和死亡率均呈现持续攀升的趋势。其中,转移作为癌症患者预后不良的重要因素,仍然是恶性肿瘤患者的主要死因之一。目前,针对肿瘤转移的临床治疗手段主要是化学治疗,然而市场上大多数化疗药物都存在一定的毒副作用。长期的化疗会诱导癌细胞产生耐药性,将进一步降低化疗药物在肿瘤临床治疗中的效果。海洋微生物多糖因其具有生物相容性好,低毒以及容易获得等优势,已经成为海洋科学研究的重点之一,能够为研发高效低毒的抗肿瘤转移新药提供候选化合物。EPS11是我们实验室前期发现的一种有抗肿瘤效果的海洋细菌多糖,具有潜在的抗肿瘤转移的潜力,但相应的分子机制并不清晰。本论文结合蛋白质组学、细胞生物学等手段从体外和体内两个层次详细研究了EPS11抗肿瘤转移的分子机制,为开发多糖类抗肿瘤转移药物提供了可靠的理论依据和药物前体。

     本研究首先通过MTT法检测EPS11对多种肝癌细胞的毒性,表明其能够浓度和时间依赖性地抑制肝癌细胞Huh7.5、Bel-7402和HepG2的增殖。同时,利用光学显微镜观察发现EPS11能够引起肝癌细胞Huh7.5、Bel-7402和HepG2的聚集成团、脱黏附。进而,我们通过结晶紫染色法定量检测了EPS11对肝癌细胞黏附率的影响,并利用扫描电镜(SEM)观察了EPS11对细胞表面结构的作用。结果显示,EPS11能够浓度和时间依赖性地降低肝癌细胞的黏附能力,破坏肝癌细胞表面的伪足状结构。细胞黏附率的降低与细胞伪足状结构数量的减少相一致,表明细胞表面伪足状结构在肝癌细胞黏附方面扮演着重要的角色。为了深入探究EPS11抑制肿瘤细胞转移的功能,本研究进一步利用划痕和Transwell实验证明了EPS11能够通过降低肝癌细胞Huh7.5的黏附能力并破坏其表面结构而抑制肝癌细胞Huh7.5的迁移。

    本研究通过蛋白质组学全面分析了EPS11对肝癌细胞Huh7.5生物学的进程影响,进一步揭示EPS11抑制肝癌细胞Huh7.5生长增殖和转移的机制。通过数据分析发现EPS11作用Huh7.5细胞后,大量细胞黏附分子的表达水平显著下调。其中,细胞黏附分子CD99下调最为显著。由于CD99在细胞增殖、黏附、迁移以及分化等方面均发挥重要的作用,我们将其作为EPS11作用的关键效应分子进行深入研究。通过实时荧光定量PCR(qRT-PCR)和Western blotting对蛋白质组的结果进行验证,发现EPS11在mRNA和蛋白水平上均能够显著下调CD99的表达,尤其是在蛋白水平。进而我们在肝癌细胞Huh7.5中过表达CD99分子,并检测此时EPS11对肝癌细胞Huh7.5增殖率、黏附能力和迁移能力的变化,进而验证CD99在EPS11抑制肝癌细胞Huh7.5增殖和迁移进程中的重要性。结果表明,过表达CD99能够部分逆转EPS11引起的肝癌细胞Huh7.5增殖率、黏附能力和迁移能力的降低,进一步证实CD99在EPS11的抗肝癌转移机制中发挥着重要作用,同时也为CD99作为肝癌靶向治疗的重要标志分子提供了证据。

    为进一步探究EPS11在体内抑制肿瘤细胞转移的效果,本研究采用黑色素瘤细胞肺转移小鼠模型进行验证。与对照组相比,注射了EPS11后能够显著减少小鼠体内黑色素瘤细胞的肺部转移灶,但并不影响小鼠的体重等生长指标,证实了EPS11在动物体内具有良好的抗肿瘤转移的作用效果,是一个潜在的抗肿瘤转移候选药物。

Other Abstract

  Cancer, as one of the major public health problems in the world, has seriously threatened human life and health, and its morbidity and mortality rates continue to rise. Metastasis, as the main factor of poor prognosis in cancer patients, is still one of the main causes of death in patients with malignant tumors. Nowadays, the clinical treatment for tumor metastasis is mainly through chemotherapy. But most of the chemotherapy drugs used in the market have certain toxic side effects. Long term chemotherapy can induce cancer cells to produce drug resistance, which will further reduce the effectiveness of chemotherapy drugs in the clinical treatment of tumors. Marine microbial polysaccharides have become one of the key points in marine scientific research because of their good biocompatibility, low toxicity and easy to get, which can provide candidate compounds for the development of high efficiency and low toxicity new drugs for anti-tumor metastasis. EPS11 is a marine bacterial exopolysaccharide found by our laboratory in the early stage, which has an anti-tumor effect. It has potential anti-tumor metastasis effect, but the corresponding molecular mechanism is not clear. In this paper, the molecular mechanism of EPS11 anti-tumor metastasis was studied in detail from in vitro and in vivo two levels by proteomics and cell biology, which provided a reliable theoretical basis and drug precursor for the development of polysaccharide anti-tumor metastatic drugs.

  In this study, MTT assay was used to detect the cytotoxic effects of EPS11 on hepatocellular cancer cells Huh7.5, Bel-7402 and HepG2, and the results indicate that EPS11 could inhibit the growth and proliferation of hepatocellular carcinoma cells in a dose- and time-dependent manner. At the same time, optical microscope observation showed that EPS11 could cause the aggregation and detachment in hepatocellular carcinoma cells. Furthermore, the effects of EPS11 on the adhesion rate and cell surface structure of hepatocellular carcinoma cells Huh7.5, Bel-7402 and HepG2 were detected by crystalline purple in quantity, scanning electron microscope (SEM), respectively. The results showed that EPS11 could both reduce the adhesion ability and destroy the cell filiform structure on the surface of hepatocellular carcinoma cells Huh7.5, Bel-7402 and HepG2 in a dose- and time-independent manner. We found that the decrease of cell adhesion rate was consistent with the decrease of cell filiform structure, which indicated that the cell filiform structure of cell surface plays an important role in the adhesion of hepatocellular carcinoma cells. In order to further explore the function of EPS11 anti-tumor metastasis, we used wound healing assay and Transwell Boyden chamber assay to prove the fact that EPS11 could inhibit the migration of hepatocellular carcinoma cells Huh7.5 by reducing its adhesion ability and destroying their surface structure.

  In this study, we used proteomics technology to reveal the mechanism of EPS11 on the biological process of hepatocellular carcinoma cells Huh7.5, such as inhibiting the growth, proliferation and metastasis of hepatocellular carcinoma cells. According to the data, we found that the expression levels of mainly cell adhesion molecules were significantly decreased in hepatocellular carcinoma cells Huh7.5 after EPS11 treatment. Among them, the cell adhesion molecule CD99 was most obviously decreased. CD99 plays an important role in cell proliferation, adhesion, migration and differentiation, so we select it as a key effector molecule to deeply study the role of EPS11. After verifying it with quantitative real-time PCR (qRT-PCR) and Western blotting, we found that EPS11 could significantly reduce the expression of CD99 at mRNA and protein levels, especially in protein level. In turn, we overexpress CD99 molecules in hepatocellular carcinoma cells Huh7.5 to detect the effects of EPS11 on the proliferation, adhesion and mobility, and which were aimed to verify the importance of CD99 in EPS11 inhibiting the proliferation and migration ability of hepatocellular carcinoma cells Huh7.5. The results showed that CD99 could significantly rescued the proliferative rate, adhesion ability and migration ability decrease of hepatocellular carcinoma cells Huh7.5 after EPS11 treatment, which further confirmed that CD99 played an important role in the mechanism of anti-hepatocellular carcinoma metastasis for EPS11. It also provides evidence that CD99 can be an important marker for the target treatment of hepatocellular carcinoma.

  In order to further explore the effect of EPS11 inhibition on tumor cell metastasis in vivo, the model of pulmonary metastasis of melanoma cells was used to verify the result. Compared with the control group, the pulmonary metastasis of melanoma tumor lung colonization in mice could be significantly reduced after injection of EPS11, but it has no effect on the growth indicators in mice such as weight. It is proved that EPS11 has a good anti-tumor metastasis effect in animals and can be a potential anti-tumor metastasis candidate drug.

MOST Discipline Catalogue理学::海洋科学
Language中文
Document Type学位论文
Identifierhttp://ir.qdio.ac.cn/handle/337002/156800
Collection实验海洋生物学重点实验室
Recommended Citation
GB/T 7714
王菊. 海洋细菌胞外多糖EPS11抗肿瘤转移机制研究[D]. 中国科学院海洋研究所. 中国科学院大学,2019.
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