N-乙酰氨基葡萄糖转移酶V在卵巢癌细胞中的生物学功能及其机制研究

发布时间：2018-03-01 14:32

本文关键词： N-乙酰氨基葡萄糖转移酶V 卵巢癌增殖迁移侵袭　出处：《第二军医大学》2017年硕士论文　论文类型：学位论文

【摘要】：研究目的卵巢癌是死亡率最高的女性生殖系统恶性肿瘤,发病隐匿,其发病率和死亡率近年来有逐渐上升的趋势。由于缺乏特异性症状及有效的检查手段,大多数患者诊断时已处于卵巢癌晚期,且常已发生盆腹腔转移,给治疗带来很大的困难。尽管近年来手术和化疗药物都有所改进,但晚期卵巢癌患者的5年生存率仍然非常低。因此,探索卵巢癌的早期诊断方法和新的治疗靶点是当前研究的热点。N-糖基化是蛋白质翻译后修饰最常见的方式之一,与肿瘤细胞的各种生物学功能息息相关。N-糖基化的进程由多种不同的糖基转移酶催化,其中研究最多的是N-乙酰氨基葡萄糖转移酶V(Gn T-V)。研究发现Gn T-V在许多肿瘤的增殖、侵袭和转移中发挥重要的作用,但其在卵巢癌中的功能尚不明确。本研究主要探讨Gn T-V对卵巢癌细胞系A2780和SKOV3的增殖、迁移及侵袭等生物学行为的影响,并初步探讨其作用机制,为卵巢癌的早期诊断与治疗靶点研究提供理论依据。研究方法分别构建干扰与过表达Gn T-V的慢病毒载体,转染卵巢癌细胞系A2780和SKOV3,转染成功后通过RT-PCR技术检测病毒干扰/过表达效率,并通过细胞增殖实验(CCK-8法)、细胞划痕实验、Transwell实验及流式细胞术来观察Gn T-V的干扰/过表达对卵巢癌细胞的增殖、迁移及侵袭等生物学行为的影响。利用Western blot检测Gn T-V的干扰/过表达后卵巢癌细胞内E-cadherin、laminin、MMP-2和MMP-9等蛋白的变化,初步探讨Gn T-V影响卵巢癌细胞生物学行为的作用机制。研究结果用慢病毒干扰了Gn T-V的表达后,RT-PCR结果显示A2780和SKOV3细胞内Gn T-V的表达明显下降,CCK-8法检测发现A2780和SKOV3细胞的增殖能力增强,划痕实验发现A2780和SKOV3细胞的迁移能力增强,Transwell实验发现A2780和SKOV3细胞的侵袭能力增强,流式细胞术发现A2780和SKOV3细胞的细胞周期得到促进,Western blot结果显示Gn T-V的表达降低后,A2780和SKOV3细胞内的E-cadherin的表达水平下降,而MMP-2及MMP-9的表达增加。反之,过表达Gn T-V后,A2780和SKOV3细胞的增殖、迁移和侵袭能力都明显减弱,同时细胞内的E-cadherin的表达水平增强,laminin、MMP-2及MMP-9的表达下降。结论卵巢癌细胞中Gn T-V的表达能抑制细胞的增殖、迁移和侵袭能力,其生物学功能可能与上调E-cadherin的表达及下调laminin、MMP-2与MMP-9的表达有关。
[Abstract]:The purpose of the study of ovarian cancer is the highest mortality of female genital malignant tumors, the incidence of occult, the incidence and mortality of the trend has gradually increased in recent years. Due to the lack of specific symptoms and effective means of inspection, the majority of patients are in advanced ovarian cancer, and often have abdominal metastasis, which brought great difficulties to although in recent years the treatment. Surgery and chemotherapy drugs have been improved, but the 5 year survival rate of patients with advanced ovarian cancer is still very low. Therefore, to explore the method of early diagnosis of ovarian cancer and new therapeutic targets is a hotspot of current research of.N- glycosylation is one of the most common protein post-translational modifications, and various the biological function of tumor cells is closely related to the.N- glycosylation process of enzyme catalyzed by different glycosyl transfer, most of them are N- N-acetylglucosaminyltransferase V (Gn T-V). The discovery of Gn T-V proliferation in many tumors, play an important role in invasion and metastasis in ovarian cancer, but its function is not clear. This study focused on Gn T-V and SKOV3 A2780 on the proliferation of ovarian cancer cell lines, the influence of biological behavior of migration and invasion, and to explore its mechanism. For ovarian cancer early diagnosis and treatment and provide a theoretical basis for the research target. Research methods were constructed interference and overexpression of Gn T-V lentiviral vector was transfected into ovarian cancer cell lines A2780 and SKOV3, after the success of transfection by detecting virus interference / RT-PCR expression efficiency, and by cell proliferation assay (CCK-8 method), cell the scratch test, interference / Transwell assay and flow cytometry was used to observe the Gn expression of T-V in ovarian cancer cell proliferation, migration and invasion influence the biological behavior of the Western / blot. The interference detection Gn T-V table As ovarian cancer cells E-cadherin, laminin, MMP-2 and MMP-9 protein in the preliminary mechanism of Gn T-V influence the biological behavior of ovarian cancer cells. The results of the study by lentiviral expression of Gn after T-V, RT-PCR showed that the expression of A2780 and SKOV3 cells in Gn T-V decreased significantly, CCK-8 detecting method enhanced A2780 and SKOV3 cell proliferation, scratch test showed enhanced migration of A2780 and SKOV3 cells, Transwell showed enhanced A2780 and SKOV3 cell invasion ability, found that A2780 and SKOV3 can promote the cell cycle by flow cytometry, Western blot showed decreased expression of Gn after T-V, the expression level of A2780 and SKOV3 of E-cadherin cells decreased, while the expression of MMP-2 and MMP-9 increased. On the other hand, over expression of Gn after T-V, A2780 and SKOV3 cell proliferation, migration and invasion were significantly reduced, with the The expression level of E-cadherin cells in the enhancement of laminin, decreased expression of MMP-2 and MMP-9. Conclusion the expression of Gn in ovarian cancer cell T-V can inhibit cell proliferation, migration and invasion, the expression of its biological function may be related to the upregulation of E-cadherin and downregulation of laminin, the expression of MMP-2 and MMP-9.

【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】：R737.31

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