林淑萍臺灣大學：醫事技術學研究所安鳳鳴An, Feng-MingFeng-MingAn2007-11-292018-07-062007-11-292018-07-062004http://ntur.lib.ntu.edu.tw//handle/246246/62833過去發現乳癌的病人體內動情素的上升會使得癌細胞增生，所以科學家利用動情素的衍生物競爭性的結合動情素接受體，藉以達到治療的效果。然而有些乳癌病人的癌細胞上卻缺乏動情素接受體，使得這樣的病灶更難以治療。於是科學家轉而尋覓其他治療標的，NF-kB便是其中之一。正常狀態下，細胞中的NF-kB處於未活化的狀態，而乳癌細胞中NF-kB處於持續性的活化狀態。 於是在本篇研究中，設計了NF-kB decoy -短片段雙股寡核苷酸序列-可以與NF-kB結合，藉以抑制乳癌細胞中NF-kB的活化，進一步的達到抑制乳癌細胞的生長以及癌細胞轉移的現象。方法上，利用化學合成的方式製作出NF-kB decoy，並用EMSA確定其確實可以和NF-kB結合，並可以抑制NF-kB的活化。後續利用ACP assay發現NF-kB decoy具有抑制乳癌細胞生長的能力。 癌轉移是一種多階段性的病程，包含了invasion、migration、trafficking而至metastasis的過程。所以在研究上，利用pre-treated NF-kB decoy的乳癌細胞進行cell adhesion assay和migration assay的試驗。初步的結果顯示NF-kB decoy對於cell adhesion以及cell migration的能力有顯著的抑制效果。 後續的實驗上針對受到NF-kB影響相關於cell adhesion及migration之間信息傳遞中主要的蛋白變化做分析，過去已知PI3K pathway和cell migration息息相關。而針對cell invasion，MMP-9在promoter的位置具有NF-kB結合的位置，MMP-2在過去證實受到SDF-1的刺激後會增加。實驗上利用gelatin zymography以及western blot做為分析工具，藉以觀察NF-kB decoy抑制乳癌細胞的情形。Numerous experimental data and clinical studies have established that estrogen plays a major role in the initiation and progression of breast cancers. Thus antiestrogens were used but did not work on estrogen receptor (ER)-negative breast cancer cells. Another therapeutic target we are interested in is NF-kB. NF-kB was expressed constitutively in ER-negative breast cancer cells and might affect cell growth and metastasis. Thus inhibition of NF-kB is a plausible way to develop anti-cancer drugs. Therefore, our strategy is using synthetic oligonucleotides containing NF-kB binding sequence, which is 21 bps in length and is modified at both ends with phosphorothioate linkages. This NF-kB decoy demonstrates NF-kB binding activity and can be easily measured by electrophoresis mobility shift assay (EMSA). Our data showed that transfection of NF-kB decoy to ER-negative breast cancer cell line, MDA-MB-231, significantly block NF-kB activation. To confirm the transfection efficiency, we used fluorescein-labeled NF-kB decoy (F-decoy) and analyzed by flow cytometry and fluorescence microscopy. For cell viability, we used ACP assay to measure the living cells after treating NF-kB decoy or not. Our results showed that relatively high concentration was needed to cause significant inhibition of cell growth than inhibition of cell migration. On the other hand, it is found that NF-kB decoy significantly inhibits adhesion and migration of MDA-MB-231 cells. Whereas NF-kB cause widely effects on cell adhesion, invasion, and migration, we focused on the interaction of CXCR4 and SDF-1Chinese abstract (摘要) English abstract Abbreviations 1. Introduction 9 2. Materials 18 Methods 28 3. Results 37 4. Discussions 42 Figures 49 References 60 Appendixes 711337635 bytesapplication/pdfen-US乳癌雙股寡核酸轉移MetastasisBreast CancerDecoy[SDGs]SDG3otherhttp://ntur.lib.ntu.edu.tw/bitstream/246246/62833/1/ntu-93-R91424018-1.pdf