DEK在乳腺癌细胞向内皮细胞转化中的功能与分子机制研究

发布时间：2018-05-01 18:46

本文选题：DEK + nanog　；参考：《青岛大学》2017年硕士论文

【摘要】：DEK蛋白广泛存在于细胞核内,可磷酸化,同时它也是一种染色质架构蛋白。本实验旨在研究DEK调控乳腺癌细胞向内皮细胞转化中的功能与分子机制。目的:研究DEK在乳腺癌细胞向乳腺癌干细胞转化进而分化成内皮细胞过程中的作用及分子机制,进而明确肿瘤细胞生长、分化的具体分子机制,为乳腺癌的治疗提供新的依据。方法:将实验室保存的DEK质粒在ZR75-1及MCF-7中建立稳定克隆的细胞株,通过流式细胞分选术在上述已建立的稳定克隆细胞系中检测CD44+/CD24-细胞的含量,分选得到CD44+/CD24-的细胞。用干细胞培养基进行培养,1-2周后观察乳腺微球体的形成。利用荧光实时定量PCR(RT-PCR)及western blot(WB)对细胞系中可能影响DEK表达的干性基因(c-myc、klf4、OCT4、nanog、Notch-2等)进行初步的筛选,然后在m RNA及蛋白质水平进行进一步的验证。利用免疫荧光(IF),荧光实时定量PCR(RT-PCR)及western blot(WB)技术检测细胞系中内皮细胞表面标志物CD31、CD144、VWF及VEGFR2的表达。利用Matrigel胶对细胞系进行体外培养,观察脉管样结构的形成。通过合成nanog的si RNA进行RTPCR,western blot,免疫荧光及Matrigel胶培养等实验,观察乳腺球、脉管样结构形成、内皮细胞表面marker的变化。结果:(1)通过流式细胞分选术发现在高表达DEK稳定细胞系中干细胞的含量明显升高。用干细胞悬浮培养CD44+/CD24-的细胞能形成乳腺微球体。(2)荧光实时定量PCR及western blot等技术发现在DEK稳定细胞系中干性基因nanog的含量较其它干性基因明显升高。(3)利用免疫荧光,荧光实时定量RT-PCR及western blot对内皮细胞表面标志物CD31、CD144、VEGFR2、VWF等进行检测,发现均明显升高。经Matrigel胶进行培养,能形成脉管样的结构。(4)免疫荧光(IF),荧光实时定量PCR(RT-PCR)及western blot(WB)实验结果显示,高表达DEK稳定细胞系中转入合成nanog的si RNA后,内皮细胞表面标志物的表达明显降低,经Matrigel胶培养后,未观察到脉管样结构的形成。结论:(1)高表达DEK细胞系中干细胞含量明显增加。(2)在DEK稳定细胞系中干性基因nanog调控乳腺癌细胞向乳腺癌干细胞转化。(3)高表达DEK乳腺癌细胞系中内皮细胞表面标志物明显升高。(4)高表达DEK稳定细胞系中转入nanog的si RNA后,内皮细胞表面标志物的表达显著降低,脉管样结构消失,初步证实DEK通过上调nanog介导乳腺癌细胞向内皮细胞转化。意义:本研究证实了DEK能够促进乳腺癌细胞转化内皮细胞,并且其分子机制是通过上调nanog实现的。为乳腺癌的治疗提供新的实验依据,给乳腺癌患者带来新的希望。
[Abstract]:DEK protein is widely present in the nucleus and phosphorylated, and it is also a chromatin framework protein. The aim of this study was to investigate the function and molecular mechanism of DEK in regulating the transformation of breast cancer cells to endothelial cells. Objective: to study the role and molecular mechanism of DEK in the process of transforming breast cancer cells into breast cancer stem cells and then differentiate into endothelial cells, and then clarify the specific molecular mechanism of tumor cell growth and differentiation, and provide a new basis for the treatment of breast cancer. Methods: the stable cloned cell lines were established in ZR75-1 and MCF-7 by using the DEK plasmids preserved in the laboratory. The content of CD44 / CD24- cells was detected by flow cytometry, and the CD44 / CD24- cells were isolated. The formation of mammary microspheres was observed after 1-2 weeks of culture on stem cell culture medium. PCR RT PCR and western blotWB) were used to screen the dry gene, c-myclf4OCT4OCT4, Notch-2, which might affect the expression of DEK in the cell line. The results were further verified at the level of m RNA and protein. The expression of endothelial cell surface marker CD31, CD144, VWF and VEGFR2 was detected by immunofluorescence, real-time quantitative PCR RT-PCR and western blotWB technique. The cell line was cultured in vitro with Matrigel gel to observe the formation of vascular-like structure. Si RNA of nanog was synthesized by RT PCR western blot, immunofluorescence and Matrigel gel culture to observe the formation of mammary gland ball, vascular like structure and marker on endothelial cell surface. Results (1) flow cytometry showed that the content of stem cells in stable cell lines with high expression of DEK was significantly increased. Fluorescence real-time quantitative PCR and western blot techniques showed that the content of dry gene nanog in DEK stable cell line was significantly higher than that of other dry genes. Fluorescence real-time quantitative RT-PCR and western blot were used to detect the endothelial cell surface marker CD31, CD144, VEGFR2VWF and VWF. The results of Matrigel gel culture showed that the vascular-like structure, I. e. 4) immunofluorescence, fluorescence real time quantitative PCRRT PCR and western blotWB) showed that the expression of endothelial cell surface markers in stable cell lines with high expression of DEK was significantly decreased after being transferred to si RNA which synthesized nanog. After cultured with Matrigel gel, no vascular-like structure was observed. ConclusionThe stem cell content in DEK cell line with high expression of DEK increased significantly. (2) in DEK stable cell line, the dry gene nanog regulated the transformation of breast cancer cells to breast cancer stem cells. 3) the endothelial cell surface markers in DEK breast cancer cell line were overexpressed. The expression of si RNA in DEK stable cell line was significantly higher than that in nanog stable cell line. The expression of endothelial cell surface markers was significantly decreased and vascular structure disappeared. It was preliminarily confirmed that DEK mediated the transformation of breast cancer cells to endothelial cells through up-regulation of nanog. Significance: this study demonstrated that DEK can promote the transformation of breast cancer cells into endothelial cells, and its molecular mechanism is through up-regulation of nanog. To provide a new experimental basis for the treatment of breast cancer, and bring new hope to breast cancer patients.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.9

【参考文献】