【摘要】：[目的]:本文通過外加TGF-β1,誘導(dǎo)乳腺癌細(xì)胞MCF-7從上皮細(xì)胞向間充質(zhì)轉(zhuǎn)化(epithelial-mesenchymal transition, EMT)。通過建立穩(wěn)定的 EMT 細(xì)胞模型,探究MCF-7發(fā)生EMT轉(zhuǎn)化過程中能量代謝方式的變化及其調(diào)控機(jī)制。[方法]:用含有TGF-β1 (2ng/ml)的培養(yǎng)基培養(yǎng)MCF-7細(xì)胞9天,之后換正常培養(yǎng)基培養(yǎng)9天。在這個(gè)過程中MCF-7細(xì)胞完成了從上皮細(xì)胞向間質(zhì)細(xì)胞轉(zhuǎn)化,再?gòu)拈g質(zhì)細(xì)胞轉(zhuǎn)化成上皮細(xì)胞的過程。實(shí)驗(yàn)中,我們用qPCR和western檢測(cè)了 EMT上皮細(xì)胞標(biāo)志蛋白E-耗黏蛋白(E-cadherin)、間質(zhì)細(xì)胞標(biāo)志蛋白波形蛋白(Vimentin)和轉(zhuǎn)錄因子Snail 1的表達(dá),同時(shí)通過細(xì)胞侵襲實(shí)驗(yàn)檢測(cè)了細(xì)胞侵襲能力的變化;用western檢測(cè)了糖代謝相關(guān)蛋白PDH、LDH、NDUFB8、TFAM和COX1的表達(dá)變化;通過流式細(xì)胞術(shù)和qPCR檢測(cè)了線粒體數(shù)量以及線粒體DNA拷貝數(shù);用qPCR和western檢測(cè)了 FASN、CPT1和CD36等與脂代謝相關(guān)基因或蛋白的表達(dá);并檢測(cè)了可能引起脂代謝變化的相關(guān)信號(hào)通路蛋白的表達(dá)變化;最后我們收集到20例乳腺癌臨床樣本,利用免疫組化方法檢測(cè)了脂代謝相關(guān)蛋白在乳腺癌組織中的表達(dá)部位和表達(dá)水平的變化。通過開展上述研究,我們得到如下研究結(jié)果:[結(jié)果](1)成功構(gòu)建了 TGF-β1誘導(dǎo)MCF-7從上皮細(xì)胞向間質(zhì)細(xì)胞的轉(zhuǎn)化,轉(zhuǎn)化過程中觀察到相應(yīng)的細(xì)胞形態(tài)學(xué)的變化和粘附性降低,同時(shí)檢測(cè)到E-cadherin表達(dá)降低,Vimentin和Snail1表達(dá)量升高。(2)MCF-7在發(fā)生EMT過程中細(xì)胞增殖能力降低,但ATP水平增高,細(xì)胞侵襲能力增加。(3)MCF-7在發(fā)生EMT過程中氧化磷酸化相關(guān)蛋白NDUFB8、TFAM和COX1表達(dá)顯著增加,線粒體數(shù)量和線粒體DNA也明顯增加。表明EMT轉(zhuǎn)化過程中細(xì)胞線粒體氧化磷酸化活性增強(qiáng)。(4) EMT轉(zhuǎn)化過程中FASN表達(dá)降低,CPT-1和CD36表達(dá)升高,細(xì)胞脂肪酸的合成途徑降低脂肪酸β氧化增強(qiáng)。(5) TGF-β1處理MCF-7后檢測(cè)到P-AMPK表達(dá)增高而ACC的表達(dá)降低,P-AMPK可能通過抑制ACC表達(dá)從而增強(qiáng)CPT-1的表達(dá),增加細(xì)胞內(nèi)脂肪酸氧化。(6)對(duì)20例乳腺癌臨床腫瘤樣本進(jìn)行免疫組化分析發(fā)現(xiàn)在乳腺癌腫瘤組織中E-cadherin高表達(dá)的上皮細(xì)胞中FASN高表達(dá),而在Vimentin高表達(dá)的間質(zhì)細(xì)胞中CPT-1α高表達(dá),表明在乳腺癌組織中存在上皮細(xì)胞和間質(zhì)細(xì)胞的明顯分區(qū),在上皮細(xì)胞中脂肪酸合成加強(qiáng)利于脂肪儲(chǔ)存,而間質(zhì)細(xì)胞中加強(qiáng)脂肪酸β氧化,產(chǎn)生大量ATP用于細(xì)胞侵襲和轉(zhuǎn)移。提示腫瘤組織的不同區(qū)域和不同細(xì)胞類型代謝方式不同。[結(jié)論]:TGF-β1誘導(dǎo)MCF-7發(fā)生EMT的過程中細(xì)胞增殖減慢,細(xì)胞侵襲能力增加,ATP水平增加,線粒體氧化磷酸化活性增強(qiáng),脂肪酸β氧化增加,脂肪酸合成降低。同時(shí)乳腺癌腫瘤組織中也檢測(cè)到了上皮細(xì)胞中脂肪酸合成加強(qiáng),利于脂肪儲(chǔ)存,而間質(zhì)細(xì)胞中脂肪酸β氧化加強(qiáng),產(chǎn)生大量ATP利于細(xì)胞侵襲和轉(zhuǎn)移。提示腫瘤組織的不同區(qū)域和不同細(xì)胞類型代謝方式不同。本研究對(duì)腫瘤轉(zhuǎn)移過程中的代謝特征有新的認(rèn)識(shí),為EMT轉(zhuǎn)化過程中能量代謝與轉(zhuǎn)移的關(guān)系研究奠定基礎(chǔ)。
[Abstract]:[Objective] To induce the epithelial-mesenchymal transition (EMT) of breast cancer cell line MCF-7 by adding TGF-beta 1. To establish a stable EMT cell model and explore the changes of energy metabolism and its regulation mechanism during the EMT transformation of MCF-7. MCF-7 cells were cultured in medium for 9 days, then in normal medium for 9 days. During this process, MCF-7 cells completed the transformation from epithelial cells to mesenchymal cells, and then from mesenchymal cells to epithelial cells. The expression of Vimentin and Snail-1 was detected by cell invasion assay; the expression of PDH, LDH, NDUFB8, TFAM and COX1 were detected by Western blot; the number of mitochondria and the number of mitochondrial DNA copies were detected by flow cytometry and qPCR; and the number of mitochondrial DNA copies was detected by qPCR and qPCR. Western detected the expression of FASN, CPT1 and CD36 genes or proteins related to lipid metabolism; detected the expression of signal pathway proteins that may cause lipid metabolism changes; finally, we collected 20 breast cancer clinical samples and detected the expression of lipid metabolism related proteins in breast cancer tissues by immunohistochemical method. The results were as follows: (1) TGF-beta 1 was successfully constructed to induce the transformation of MCF-7 from epithelial cells to mesenchymal cells. The morphological changes and adhesion of MCF-7 cells were observed during the transformation, and the expression of E-cadherin, Vimentin and Snail 1 were decreased. (3) The expression of oxidative phosphorylation-related proteins NDUFB8, TFAM, COX1, mitochondrial DNA and mitochondrial DNA increased significantly during EMT. Phosphorylation activity was enhanced. (4) The expression of FASN decreased, the expression of CPT-1 and CD36 increased, and the synthesis pathway of cellular fatty acid decreased the oxidation of fatty acid beta. (5) After MCF-7 was treated with TGF-beta 1, the expression of P-AMPK increased and the expression of ACC decreased. P-AMPK may enhance the expression of CPT-1 and increase intracellular lipid by inhibiting the expression of ACC. Fatty acid oxidation. (6) Immunohistochemical analysis of 20 breast cancer samples showed that FASN was highly expressed in E-cadherin-overexpressed epithelial cells, while CPT-1a was highly expressed in Vimentin-overexpressed stromal cells, suggesting that there were distinct zones of epithelial cells and stromal cells in breast cancer tissues. Fatty acid synthesis in skin cells is beneficial to fat storage, whereas in stromal cells, fatty acid beta oxidation is enhanced, resulting in a large number of ATP for cell invasion and metastasis. In addition, ATP levels increased, mitochondrial oxidative phosphorylation activity increased, fatty acid beta oxidation increased, and fatty acid synthesis decreased. At the same time, fatty acid synthesis in epithelial cells was also detected in breast cancer tissues, which was conducive to fat storage, while fatty acid beta oxidation in stromal cells was enhanced, resulting in a large number of ATP conducive to cell invasion and metastasis. This study provides a new understanding of the metabolic characteristics of tumor metastasis and lays a foundation for the study of the relationship between energy metabolism and metastasis in the process of EMT transformation.
【學(xué)位授予單位】：昆明醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R737.9

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 Liem Minh Phan;Sai-Ching Jim Yeung;Mong-Hong Lee;;Cancer metabolic reprogramming: importance, main features, and potentials for precise targeted anti-cancer therapies[J];Cancer Biology & Medicine;2014年01期

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本文編號(hào)：2234698