本文關(guān)鍵詞： 乳腺癌 HO-1 阿霉素 化療敏感性　出處：《廣西醫(yī)科大學(xué)》2015年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：目的 研究血紅素加氧酶-1(heme oxygenase, HO-1)在調(diào)控乳腺癌細(xì)胞對(duì)阿霉素敏感性中的作用及其相關(guān)分子機(jī)制。方法 (1)PI染色結(jié)合細(xì)胞流式術(shù)測(cè)定乳腺癌MDA-MB-453和MDA-MB-231細(xì)胞對(duì)阿霉素的凋亡反應(yīng)；(2)利用Western blot檢測(cè)乳腺癌MDA-MB-453和MDA-MB-231細(xì)胞在阿霉素刺激前后HO-1蛋白的表達(dá)水平；(3)采用siRNA介導(dǎo)的RNA干擾技術(shù)阻斷阿霉素對(duì)HO-1的誘導(dǎo)表達(dá),用細(xì)胞流式術(shù)檢測(cè)其對(duì)細(xì)胞凋亡的影響；(4)采用細(xì)胞自噬檢測(cè)試劑盒(ENZO)染色,并分別應(yīng)用共激聚焦顯微鏡、細(xì)胞流式術(shù)檢測(cè)細(xì)胞自噬水平；(5)應(yīng)用雙熒光素酶基因檢測(cè)、Western blot等方法檢測(cè)阿霉素刺激STAT3等轉(zhuǎn)錄因子及Src等蛋白激酶的活性變化；(6)應(yīng)用siRNA干擾、Western blot等技術(shù)探討阿霉素誘導(dǎo)HO-1表達(dá)及其影響乳腺癌細(xì)胞對(duì)阿霉素敏感性的分子機(jī)制。結(jié)果 阿霉素(0.2μM)刺激48小時(shí)后,MDA-MB-453細(xì)胞凋亡率明顯上升(從1.5%到40%),而MDA-MB-231細(xì)胞的凋亡率較低(從3.29%到13.7%)。Western blot結(jié)果顯示MDA-MB-231細(xì)胞經(jīng)阿霉素刺激后,HO-1蛋白出現(xiàn)了顯著的誘導(dǎo)表達(dá)上調(diào),而在MDA-MB-453細(xì)胞中并未觀察到此現(xiàn)象。沉默HO-1基因表達(dá)后,MDA-MB-231細(xì)胞凋亡率顯著上升。進(jìn)一步研究發(fā)現(xiàn),阿霉素可誘導(dǎo)MDA-MB-231細(xì)胞的自噬水平明顯升高,抑制自噬后MDA-MB-231細(xì)胞凋亡率明顯上升。抑制HO-1基因表達(dá)后,MDA-MB-231細(xì)胞的自噬水平顯著下降。阿霉素的刺激作用下,MDA-MB-231細(xì)胞出現(xiàn)了STAT3和磷酸化STAT3蛋白表達(dá)上調(diào),雙熒光素酶報(bào)告基因表達(dá)分析顯示STAT3的轉(zhuǎn)錄活性增強(qiáng),抑制STAT3的表達(dá)后H0-1蛋白的誘導(dǎo)表達(dá)及細(xì)胞自噬水平均顯著被抑制。在MDA-MB-231細(xì)胞中,阿霉素還顯著誘導(dǎo)Src活化水平上調(diào),抑制Src的表達(dá)后STAT3轉(zhuǎn)錄活性、H0-1蛋白及自唆表達(dá)水平均明顯下調(diào)。結(jié)論 阿霉素特異性誘導(dǎo)乳腺癌MDA-MB-231細(xì)胞HO-1高表達(dá),阻斷HO-1的誘導(dǎo)表達(dá)能顯著增加其對(duì)阿霉素的敏感性;HO-1維持乳腺癌細(xì)胞對(duì)阿霉素的抗性作用可能是通過(guò)誘導(dǎo)細(xì)胞自嚼來(lái)介導(dǎo)的;阿霉素誘導(dǎo)HO-1表達(dá)的可能機(jī)制是通過(guò)激活Src/STAT3信號(hào)通路,進(jìn)而上調(diào)HO-1的表達(dá)水平;抑制Src/STAT3/HO-l/autophagy信號(hào)途徑的激活能顯著增加乳腺癌細(xì)胞對(duì)阿霉素的敏感性。
[Abstract]:Objective to study heme oxygenase of heme oxygenase-1. The role of HO-1 in regulating the sensitivity of breast cancer cells to adriamycin and its related molecular mechanisms. Pi staining and flow cytometry were used to detect the apoptosis of MDA-MB-453 and MDA-MB-231 cells to doxorubicin. (2) Western blot was used to detect the expression of HO-1 protein in MDA-MB-453 and MDA-MB-231 cells before and after adriamycin stimulation. (3) RNA interference mediated by siRNA was used to block the expression of HO-1 induced by adriamycin, and its effect on apoptosis was detected by flow cytometry. The level of autophagy was detected by confocal microscope and flow cytometry. (5) double luciferase gene assay was used to detect the activity of STAT3 and protein kinase such as Src stimulated by doxorubicin. Application of siRNA interference. Western blot and other techniques to investigate the expression of HO-1 induced by adriamycin and its molecular mechanism affecting the sensitivity of breast cancer cells to adriamycin. After 48 hours of stimulation. The apoptosis rate of MDA-MB-453 cells increased significantly (from 1.5% to 40). However, the apoptosis rate of MDA-MB-231 cells was lower (from 3.29% to 13.70.Western blot results showed that MDA-MB-231 cells were stimulated by adriamycin). The expression of HO-1 protein was significantly up-regulated, but not observed in MDA-MB-453 cells. The expression of HO-1 gene was silenced. The apoptosis rate of MDA-MB-231 cells was significantly increased. Further studies showed that doxorubicin could induce a marked increase in autophagy level of MDA-MB-231 cells. After inhibiting autophagy, the apoptosis rate of MDA-MB-231 cells increased significantly. After inhibiting the expression of HO-1 gene, the autophagy level of MDA-MB-231 cells decreased significantly. The expression of STAT3 and phosphorylated STAT3 protein was up-regulated in MDA-MB-231 cells, and the expression of double luciferase reporter gene showed that the transcriptional activity of STAT3 was enhanced. The expression of H0-1 protein and the level of autophagy were significantly inhibited after inhibiting the expression of STAT3. In MDA-MB-231 cells, doxorubicin also significantly upregulated the level of Src activation. The transcriptional activity of STAT3 was inhibited after the expression of Src. Conclusion doxorubicin specifically induces the overexpression of HO-1 in breast cancer MDA-MB-231 cells. Blocking the induced expression of HO-1 could significantly increase its sensitivity to adriamycin. The maintenance of HO-1 resistance to adriamycin in breast cancer cells may be mediated by inducing self-chewing. The possible mechanism of HO-1 expression induced by doxorubicin is by activating the Src/STAT3 signaling pathway and then upregulating the level of HO-1 expression. Inhibition of activation of Src/STAT3/HO-l/autophagy signaling pathway significantly increased the sensitivity of breast cancer cells to adriamycin.
【學(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：R737.9
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本文編號(hào)：1464779