活性氧影响上皮性卵巢癌转移及其机理研究

发布时间：2019-06-08 17:50

【摘要】：背景：上皮性卵巢癌（epithelial ovarian carcinoma，EOC）是来源于卵巢上皮组织的恶性肿瘤，发病率占女性生殖系统恶性肿瘤的第三位。恶性程度高，不易早期发现，75%的患者就诊时为FIGO III期或IV期，是死亡率最高的女性生殖系统肿瘤。活性氧（reactive oxygen species，ROS）是真核细胞有氧呼吸时的产物是正常细胞代谢自然产生的，由氧分子自然形成的。氧分子（molecularoxygen）通过线粒体的有氧代谢对ATP的产生是必需的。肿瘤细胞的快速生长和增殖会导致氧需的激增。同时又因消耗氧供而处于低氧环境。肿瘤细胞的改编码代谢主要依赖着糖酵解和需氧呼吸的降低。因此，氧耗促进了肿瘤细胞的永生，而且直接作用于新生血管的生长以促进肿瘤生长。大量研究表明，，ROS与肿瘤的发生发展以及转移有关1，2，包括参与上皮间充质转化（epithelial mesenchymaltransition，EMT）等复杂过程2，成为基础医学和生命科学的研究热点。但是，在卵巢癌患者中，ROS是否参与及其作用机制目前仍不清楚。E-钙粘蛋白（E-cadherin,简称E-cad）是上皮间充质改变（epithelial-mesenchymaltransition，EMT） I型标志性分子，可维持细胞间紧密连接，阻止细胞活动侵袭及转移扩散3。目前在肿瘤转移通路上，有学者提出HIF-1α及LOX可能是E-cadherin的上游分子4，5。因而本研究大胆假设并验证，HIF-1α、LOX参与ROS介导的卵巢癌间充质转化通路。目的：本研究在调节ROS水平情况下，检测相对应EOC E-cadherin水平，来研究ROS水平高低是否影响EOC EMT发生，并进一步探讨其影响机制。即ROS是否会介导HIF-1α的高表达，从而上调LOX，并通过抑制E-cadherin等介导EMT使肿瘤细胞发生上皮间充质转化，增强肿瘤细胞的侵袭能力，促进肿瘤细胞的转移,为上皮性卵巢癌的转移机制提供新的理论基础。方法：研究分为四部分进行，前两部分以人上皮性卵巢癌细胞株SKOV-3为切入点，探索ROS水平与EOC转移的相关性。并通过SiRNA基因沉默技术转染细胞、药物干预分别抑制了HIF-1α和LOX，并测定HIF-1α、LOX及E-cadherin蛋白和或mRNA表达，推测其可能的分子机制。第三部分以动物实验为着手，着重分析大黄素单药以及未予药物干预对照组小鼠间的成瘤差异，并对EMT标志分子E-cadherin进行蛋白免疫组化实验，验证活性氧与上皮间充质改变和肿瘤转移之间的关系。第四部分进行卵巢癌患者临床资料分析，并对收集的标本进行E-cadherin蛋白的免疫组化实验。结论：我们发现，临床标本的E-cadherin与卵巢癌分期关系密切。基于上述四部分实验，我们获得以下通路推测：上皮性卵巢癌ROS↑—— HIF-1α↑—— LOX↑—— E-cadherin↑——肿瘤细胞发生EMT——侵袭性↑——肿瘤转移
[Abstract]:Background: epithelial ovarian cancer (epithelial ovarian carcinoma,EOC) is a malignant tumor from ovarian epithelial tissue, which accounts for the third highest incidence of malignant tumor of female reproductive system. The malignant degree was high and it was not easy to detect early. 75% of the patients were in FIGO III stage or IV stage, which was the female reproductive system tumor with the highest mortality. Reactive oxygen species (reactive oxygen species,ROS) is the product of aerobic respiration in eukaryotic cells, which is naturally produced by normal cell metabolism and formed by oxygen molecules. Oxygen molecule (molecularoxygen) is necessary for ATP production through aerobic metabolism of mitochondria. The rapid growth and proliferation of tumor cells can lead to a surge in oxygen demand. At the same time, it is in a hypoxia environment because of the consumption of oxygen supply. The modification metabolism of tumor cells mainly depends on glycolysis and the decrease of aerobic respiration. Therefore, oxygen consumption promotes the immortality of tumor cells and directly acts on the growth of neovascularization to promote tumor growth. A large number of studies have shown that ROS is related to the occurrence, development and metastasis of tumors. 2, including participation in complex processes such as epithelial mesenchymal transformation (epithelial mesenchymaltransition,EMT), has become a hot research topic in basic medicine and life science. However, it is still unclear whether ROS is involved and its mechanism in patients with ovarian cancer. E-cadherin (E-cad) is the iconic molecule of epithelial mesenchyma change (epithelial-mesenchymaltransition,EMT) type I, which is known as E-cadherin (E cadherin, E cadherin). It can maintain the tight junction between cells and prevent the invasion and metastasis and diffusion of cell activity. 3. At present, in the tumor metastasis pathway, some scholars have suggested that HIF-1 伪 and LOX may be the upstream molecules of E-cadherin 4, 5. Therefore, this study boldly hypothesized and verified that HIF-1 伪 and LOX are involved in ROS-mediated mesenchymal transformation pathway in ovarian cancer. Objective: to detect the corresponding EOC E-cadherin level under the condition of adjusting ROS level, to study whether ROS level affects the occurrence of EOC EMT, and to further explore its mechanism. That is, whether ROS mediates the high expression of HIF-1 伪, thus upregulating LOX, and promoting the metastasis of tumor cells by inhibiting E-cadherin and other mediating EMT to transform tumor cells into epithelial mesenchyma, enhance the invasiveness of tumor cells and promote the metastasis of tumor cells. It provides a new theoretical basis for the metastasis mechanism of epithelial ovarian cancer. Methods: the study was divided into four parts. The first two parts were based on human epithelial ovarian cancer cell line SKOV-3 to explore the relationship between ROS level and EOC metastasis. The expression of HIF-1 伪 and LOX, were inhibited by SiRNA gene silencing technique, and the expression of HIF-1 伪, LOX, E-cadherin protein and mRNA were detected, and the possible molecular mechanism was speculated. In the third part, based on the animal experiment, the tumor formation difference between emodin single drug and control group without drug intervention was analyzed, and the protein Immunohistochemical test of EMT marker E-cadherin was carried out. To verify the relationship between reactive oxygen species (Ros) and epithelial mesenchymal changes and tumor metastasis. In the fourth part, the clinical data of patients with ovarian cancer were analyzed, and the collected samples were studied by immunohistochemistry of E-cadherin protein. Conclusion: E-cadherin in clinical specimens is closely related to the stage of ovarian cancer. Based on the above four parts of the experiment, we obtain the following pathway speculation: epithelial ovarian cancer ROS-HIF-1 伪-LOX-E-cadherin-tumor cells develop EMT-- invasive tumor metastasis-tumor metastasis
【学位授予单位】：上海交通大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.31

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