盐霉素抑制上皮性卵巢癌细胞发生上皮间质转化的作用及其机制研究

发布时间：2018-05-20 09:22

本文选题：上皮性卵巢癌 + 上皮间质转化　；参考：《山东大学》2016年硕士论文

【摘要】：研究背景及目的：上皮性卵巢癌是妇科恶性肿瘤中死亡率最高的肿瘤,主要原因是大多数患者确诊时已发展为疾病的晚期,伴随腹膜种植和远处转移。尽管近年来卵巢癌的治疗已经取得了很大进展,但是卵巢癌患者的高复发率和高转移率使其预后并未得到明显改善。在深入理解卵巢癌转移的分子机制的基础上,寻找可以抑制其转移的药物,对提高卵巢癌治疗的有效率、改善其预后是十分必要的。上皮间质转化(epithelial-mesenchymal transition, EMT)是一种发育调控性程序,在胚胎发育、器官形成、伤口愈合等生理过程中发挥重要作用。近年来,在乳腺癌、肝癌、卵巢癌等研究中发现,上皮性癌细胞模拟此过程后,即可获得多种侵袭和迁移的能力,提示EMT与癌症的侵袭和转移密切相关。因此,寻找阻断EMT的药物具有潜在的临床意义。盐霉素是一种单羧基聚醚类抗生素,广泛地用于鸡的抗球虫病治疗。在乳腺癌的研究中发现盐霉素能够靶向杀灭乳腺癌干细胞。EMT与细胞获得干性的密切关系提示我们EMT可能是盐霉素靶向抑制干细胞的主要作用机制。近年来,盐霉素抑制EMT的能力在肝癌、结肠癌、肺癌中被证实,但在卵巢癌中尚未见报道。本文旨在研究盐霉素对人上皮性卵巢癌细胞系A2780和SK-OV-3增殖、侵袭、迁移等能力的影响并对其作用机制进行初步的探讨。方法：CCK-8法检测不同浓度盐霉素(0,2,4,8,16μM)作用不同时间后A2780和SK-OV-3细胞的生存率。Transwell侵袭小室检测盐霉素对A2780细胞和SK-OV-3细胞侵袭和迁移能力的影响。Western-blot和RT-PCR检测盐霉素作用前后A2780和SK-OV-3细胞中上皮表型分子E-cadherin和Keratin,间质表型分子N-cadherin和Vimentin以及Wnt/β-catenin信号通路相关分子表达的变化。免疫荧光方法用来定位P-catenin在卵巢癌细胞中的分布；GSK-3β抑制剂(SB216763)用来激活卵巢癌细胞中的Wnt/β-catenin信号通路。结果：(1)盐霉素对卵巢癌细胞的增殖能力有抑制作用：应用CCK-8法检测不同浓度盐霉素(0,2,4,8,16μM)作用于A2780和SK-OV-3细胞24,48,72小时后细胞的生长率,结果显示：盐霉素对这两种细胞均有抑制增殖的作用,且呈时间依赖性和浓度依赖性。(2)盐霉素对卵巢癌细胞的侵袭和迁移能力有抑制作用：应用Transwell小室检测盐霉素作用前后A2780和SK-OV-3细胞侵袭和迁移能力的改变,结果显示：与对照组相比,盐霉素处理后的卵巢癌细胞的侵袭能力和迁移能力均明显下降(P0.001)。(3)盐霉素对卵巢癌细胞发生EMT有抑制作用：应用Western-blot和RT-PCR方法检测盐霉素对A2780和SK-OV-3细胞上皮性分子E-cadherin、Keratin和间质性分子N-cadherin、Vimentin表达的影响,结果显示：与对照组相比,经盐霉素处理的卵巢癌细胞的上皮性分子表达明显升高,而间质性分子表达明显下降,且随着药物浓度的增加变化越明显。(4)盐霉素对卵巢癌细胞中Wnt/β-catenin信号通路有阻断作用：应用Western-blot和RT-PCR方法检测盐霉素作用前后Wnt/β-catenin信号通路相关分子β-GSK-3β-ser9、β-catenin、slug、Axin2、CCND1的表达,结果显示：与对照组相比,实验组中各分子的表达随着药物浓度的增加逐渐下降；用免疫荧光方法定位盐霉素作用前后β-catenin在卵巢癌细胞中的分布,结果显示：与对照组相比,实验组中细胞内的β-catenin大部分集中于胞浆内,在胞核内的分布明显减少。(5)盐霉素抑制卵巢癌细胞发生EMT的机制研究：GSK-3β抑制剂(SB216763)处理后,Wnt/β-catenin信号通路各相关分子表达均上调,同时我们检测EMT相关蛋白发现,在两个细胞系中,上皮表型分子均表达下调且间质表型分子表达上调；我们将盐霉素作用于SB216763预处理后的细胞,发现Wnt/β-catenin信号通路各相关分子的表达较SB216763单一处理后的细胞下调。结论：(1)盐霉素通过抑制上皮性卵巢癌细胞发生EMT而抑制其侵袭和迁移能力。(2)盐霉素抑制上皮性卵巢癌细胞发生EMT是通过抑制Wnt/β-catenin信号通路完成的。
[Abstract]:Background and objective: epithelial ovarian cancer is the highest death rate in gynecologic malignancies. The main reason is that most patients have been developed into late stage of the disease, accompanied by peritoneal implantation and distant metastasis. Although the treatment of ovarian cancer has been greatly expanded in recent years, the high recurrence rate and high rotation of ovarian cancer patients On the basis of understanding the molecular mechanism of ovarian cancer metastasis, it is necessary to improve the efficiency of ovarian cancer treatment and improve the prognosis of ovarian cancer. Epithelial-mesenchymal transition (EMT) is a developmental regulatory procedure. It plays an important role in embryonic development, organ formation, wound healing and other physiological processes. In recent years, in the studies of breast cancer, liver cancer, and ovarian cancer, epithelial cancer cells mimic the ability of multiple invasiveness and migration after this process, suggesting that EMT is closely related to the invasion and metastasis of cancer. Therefore, the search for drugs to block EMT is found. There is potential clinical significance. Salt mycin is a single carboxyl polyether antibiotic which is widely used in chicken anti coccidiosis treatment. In the study of breast cancer, it is found that salt mycin can target the killing of.EMT and cells in breast cancer stem cells, suggesting that EMT may be the main mechanism of salt mycin targeting to stem cells. In recent years, the ability of salt mycin to inhibit EMT has been confirmed in liver cancer, colon cancer and lung cancer, but it has not been reported in ovarian cancer. The purpose of this study is to investigate the effect of salt mycin on the proliferation, invasion and migration of human epithelial ovarian cancer cell lines, such as proliferation, invasion and migration, and to explore the mechanism of its action by CCK-8 method. The survival rate of A2780 and SK-OV-3 cells after the action of concentration of salt mycin (0,2,4,8,16 mu M).Transwell invasion of the chamber to detect the effect of salt mycin on the invasion and migration of A2780 cells and SK-OV-3 cells.Western-blot and RT-PCR detection of the epithelial phenotype molecules E-cadherin and interstitium in A2780 and SK-OV-3 cells in A2780 and SK-OV-3 cells before and after the action of salt mycin. Changes in the expression of phenotypic molecules N-cadherin and Vimentin and Wnt/ beta -catenin signaling pathway. Immunofluorescence is used to locate P-catenin in ovarian cancer cells; GSK-3 beta inhibitor (SB216763) is used to activate the Wnt/ beta -catenin signaling pathway in ovarian cancer cells. Results: (1) the proliferation of yammycin on ovarian cancer cells Ability has inhibitory effect: CCK-8 method was used to detect the growth rate of A2780 and SK-OV-3 cells after 24,48,72 hours of different concentrations of salt mycin (0,2,4,8,16 M). The results showed that the proliferation of these two cells was inhibited by salt mycin, and it was time dependent and concentration dependent. (2) the invasion and invasion of the ovarian cancer cells by the salt mycin. The migration ability was inhibited: the changes of invasion and migration ability of A2780 and SK-OV-3 cells before and after the effect of salt mycin were detected in the Transwell chamber. The results showed that the invasiveness and migration ability of the ovarian cancer cells after salt mycin treatment were significantly lower than those in the control group (P0.001). (3) the EMT of ovarian cancer cells was inhibited by salt mycin. The effect of Western-blot and RT-PCR method was used to detect the expression of E-cadherin, Keratin and interstitial molecules N-cadherin and Vimentin in A2780 and SK-OV-3 cells, and the results showed that the expression of skin molecules in the ovarian cancer cells treated by salinomycin was significantly higher than that of the control group, and the interstitial molecular table was compared with the control group. The change was obvious with the increase of drug concentration. (4) the Wnt/ beta -catenin signaling pathway in ovarian cancer cells was blocked by salt mycin: using Western-blot and RT-PCR methods to detect the expression of Wnt/ beta -catenin signaling pathway related molecules beta -GSK-3 beta -ser9, beta -catenin, slug, Axin2, CCND1, before and after the application of Western-blot and RT-PCR methods. Compared with the control group, the expression of each molecule in the experimental group decreased gradually with the increase of drug concentration; the distribution of beta -catenin in the ovarian cancer cells before and after the immunofluorescence method showed that the intracellular beta -catenin in the experimental group was mostly concentrated in the cytoplasm and in the nucleus compared with the control group. (5) the mechanism of the inhibition of EMT in ovarian cancer cells by salt mycin: after the treatment of GSK-3 beta inhibitor (SB216763), the expression of all related molecules in the Wnt/ beta -catenin signaling pathway was up-regulated, and we detected the expression of EMT related proteins. In the two cell lines, the expression of the upper cutaneous phenotypes was down and the expression of interstitial phenotypes was expressed. The effect of salt mycin on SB216763 pretreated cells showed that the expression of Wnt/ beta -catenin signaling pathway was down regulated by SB216763 single treated cells. Conclusion: (1) salt mycin inhibits the invasion and migration of epithelial ovarian cancer cells by inhibiting the occurrence of EMT in epithelial ovarian cancer cells. (2) yammycin inhibits epithelial ovarian cancer. EMT is produced by inhibiting the Wnt/ beta -catenin signaling pathway.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R737.31

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