本文選題：DEK + nanog�。� 參考：《青島大學(xué)》2017年碩士論文

【摘要】：DEK蛋白廣泛存在于細(xì)胞核內(nèi),可磷酸化,同時(shí)它也是一種染色質(zhì)架構(gòu)蛋白。本實(shí)驗(yàn)旨在研究DEK調(diào)控乳腺癌細(xì)胞向內(nèi)皮細(xì)胞轉(zhuǎn)化中的功能與分子機(jī)制。目的:研究DEK在乳腺癌細(xì)胞向乳腺癌干細(xì)胞轉(zhuǎn)化進(jìn)而分化成內(nèi)皮細(xì)胞過程中的作用及分子機(jī)制,進(jìn)而明確腫瘤細(xì)胞生長(zhǎng)、分化的具體分子機(jī)制,為乳腺癌的治療提供新的依據(jù)。方法:將實(shí)驗(yàn)室保存的DEK質(zhì)粒在ZR75-1及MCF-7中建立穩(wěn)定克隆的細(xì)胞株,通過流式細(xì)胞分選術(shù)在上述已建立的穩(wěn)定克隆細(xì)胞系中檢測(cè)CD44+/CD24-細(xì)胞的含量,分選得到CD44+/CD24-的細(xì)胞。用干細(xì)胞培養(yǎng)基進(jìn)行培養(yǎng),1-2周后觀察乳腺微球體的形成。利用熒光實(shí)時(shí)定量PCR(RT-PCR)及western blot(WB)對(duì)細(xì)胞系中可能影響DEK表達(dá)的干性基因(c-myc、klf4、OCT4、nanog、Notch-2等)進(jìn)行初步的篩選,然后在m RNA及蛋白質(zhì)水平進(jìn)行進(jìn)一步的驗(yàn)證。利用免疫熒光(IF),熒光實(shí)時(shí)定量PCR(RT-PCR)及western blot(WB)技術(shù)檢測(cè)細(xì)胞系中內(nèi)皮細(xì)胞表面標(biāo)志物CD31、CD144、VWF及VEGFR2的表達(dá)。利用Matrigel膠對(duì)細(xì)胞系進(jìn)行體外培養(yǎng),觀察脈管樣結(jié)構(gòu)的形成。通過合成nanog的si RNA進(jìn)行RTPCR,western blot,免疫熒光及Matrigel膠培養(yǎng)等實(shí)驗(yàn),觀察乳腺球、脈管樣結(jié)構(gòu)形成、內(nèi)皮細(xì)胞表面marker的變化。結(jié)果:(1)通過流式細(xì)胞分選術(shù)發(fā)現(xiàn)在高表達(dá)DEK穩(wěn)定細(xì)胞系中干細(xì)胞的含量明顯升高。用干細(xì)胞懸浮培養(yǎng)CD44+/CD24-的細(xì)胞能形成乳腺微球體。(2)熒光實(shí)時(shí)定量PCR及western blot等技術(shù)發(fā)現(xiàn)在DEK穩(wěn)定細(xì)胞系中干性基因nanog的含量較其它干性基因明顯升高。(3)利用免疫熒光,熒光實(shí)時(shí)定量RT-PCR及western blot對(duì)內(nèi)皮細(xì)胞表面標(biāo)志物CD31、CD144、VEGFR2、VWF等進(jìn)行檢測(cè),發(fā)現(xiàn)均明顯升高。經(jīng)Matrigel膠進(jìn)行培養(yǎng),能形成脈管樣的結(jié)構(gòu)。(4)免疫熒光(IF),熒光實(shí)時(shí)定量PCR(RT-PCR)及western blot(WB)實(shí)驗(yàn)結(jié)果顯示,高表達(dá)DEK穩(wěn)定細(xì)胞系中轉(zhuǎn)入合成nanog的si RNA后,內(nèi)皮細(xì)胞表面標(biāo)志物的表達(dá)明顯降低,經(jīng)Matrigel膠培養(yǎng)后,未觀察到脈管樣結(jié)構(gòu)的形成。結(jié)論:(1)高表達(dá)DEK細(xì)胞系中干細(xì)胞含量明顯增加。(2)在DEK穩(wěn)定細(xì)胞系中干性基因nanog調(diào)控乳腺癌細(xì)胞向乳腺癌干細(xì)胞轉(zhuǎn)化。(3)高表達(dá)DEK乳腺癌細(xì)胞系中內(nèi)皮細(xì)胞表面標(biāo)志物明顯升高。(4)高表達(dá)DEK穩(wěn)定細(xì)胞系中轉(zhuǎn)入nanog的si RNA后,內(nèi)皮細(xì)胞表面標(biāo)志物的表達(dá)顯著降低,脈管樣結(jié)構(gòu)消失,初步證實(shí)DEK通過上調(diào)nanog介導(dǎo)乳腺癌細(xì)胞向內(nèi)皮細(xì)胞轉(zhuǎn)化。意義:本研究證實(shí)了DEK能夠促進(jìn)乳腺癌細(xì)胞轉(zhuǎn)化內(nèi)皮細(xì)胞,并且其分子機(jī)制是通過上調(diào)nanog實(shí)現(xiàn)的。為乳腺癌的治療提供新的實(shí)驗(yàn)依據(jù),給乳腺癌患者帶來新的希望。
[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.
【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R737.9

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