本文選題：氟維司群　切入點(diǎn)：反饋性激活效應(yīng)　出處：《天津醫(yī)科大學(xué)》2017年博士論文　論文類型：學(xué)位論文

【摘要】：研究背景及目的:內(nèi)分泌治療作為乳腺癌輔助治療重要的治療策略,能夠明顯改善激素受體陽性乳腺癌患者的預(yù)后[1],氟維司群(Fulvestrant)是常用的內(nèi)分泌藥物[2]。蛋白組學(xué)分析發(fā)現(xiàn),長(zhǎng)期雌激素剝奪細(xì)胞(long-term estrogen deprivation cells,LTED cells)的Erb B家族膜受體介導(dǎo)的PI3K/m TOR信號(hào)通路活性增強(qiáng)。證據(jù)表明,在雌激素治療耐藥的乳腺癌患者中存在PI3K/AKT信號(hào)通路的激活[3-7],從而在細(xì)胞的生長(zhǎng)增殖中發(fā)揮重要作用。然而,從目前的研究結(jié)果來看,在雌激素耐藥的細(xì)胞中,單獨(dú)應(yīng)用EGFR抑制劑吉非替尼并不能抑制AKT的反饋性激活[8]。目前的信號(hào)調(diào)控網(wǎng)絡(luò)相關(guān)研究表明腫瘤細(xì)胞生長(zhǎng)增殖及耐藥等過程十分復(fù)雜,單純的干擾或者抑制某一特定的靶點(diǎn)對(duì)于抑制細(xì)胞增殖殺滅癌細(xì)胞的作用可能十分有限。因此,如果能夠同時(shí)抑制腫瘤細(xì)胞中生長(zhǎng)相關(guān)的多個(gè)關(guān)鍵蛋白對(duì)于抑制殺滅腫瘤似乎更為有效。HSP90是細(xì)胞內(nèi)廣譜表達(dá)的伴侶蛋白,許多腫瘤細(xì)胞惡性表型的相關(guān)蛋白都依賴于HSP90的折疊功能從而發(fā)揮其生物學(xué)效應(yīng)。臨床相關(guān)資料表明,腫瘤組織中HSP90的表達(dá)相比較于癌旁正常組織要高2-3倍,而且與臨床不良預(yù)后明顯相關(guān)[9,10]。研究發(fā)現(xiàn),HSP90伴侶蛋白的高表達(dá)與乳腺癌的不良預(yù)后具有密切相關(guān),且是影響乳腺癌預(yù)后的獨(dú)立因素。HSP90的高表達(dá)與HER2陽性、ER陽性、腫瘤較大、組織學(xué)分級(jí)較高以及淋巴結(jié)轉(zhuǎn)移具有明顯相關(guān)性[11]。研究表明,HSP90能夠增強(qiáng)乳腺癌細(xì)胞對(duì)于激素治療的抵抗性,與激素治療的耐藥性相關(guān)[12]。AUY922是第二代HSP90抑制劑,它可以競(jìng)爭(zhēng)性結(jié)合于HSP90的ATP結(jié)合位點(diǎn),從而抑制HSP90的生物學(xué)活性,導(dǎo)致細(xì)胞內(nèi)很多關(guān)鍵蛋白不能被正常折疊從而失去生物學(xué)功能。由于氟維司群的反饋性激活效應(yīng)是乳腺癌細(xì)胞發(fā)生耐藥的重要原因,而且多個(gè)通路及多種通路相關(guān)蛋白參與了這一過程,然而,目前仍然沒有關(guān)于AUY922是否能夠逆轉(zhuǎn)這一反饋性激活效應(yīng)的相關(guān)研究。我們推測(cè),AUY922可能能夠通過抑制HSP90的活性,從而抑制Erb B家族及PI3K/AKT及ERK信號(hào)通路,逆轉(zhuǎn)氟維司群帶來的反饋性激活效應(yīng),為聯(lián)合用藥相關(guān)臨床試驗(yàn)提供新的基礎(chǔ)理論支持。研究方法:1.利用western blotting技術(shù)檢測(cè)了氟維司群?jiǎn)嗡幾饔脤?duì)于MCF-7和T47D細(xì)胞的ER水平的影響,以及短時(shí)間內(nèi)對(duì)于Erb B家族蛋白的反饋性激活,進(jìn)而進(jìn)一步活化下游的PI3K/AKT和ERK信號(hào)通路。并且利用RT-PCR檢測(cè)了Erb B家族的m RNA水平在氟維司群處理后的變化。2.利用western blotting技術(shù)檢測(cè)了AUY922單藥對(duì)于PI3K/AKT和ERK信號(hào)通路的抑制作用。3.Western blotting技術(shù)檢測(cè)在氟維司群處理的基礎(chǔ)上聯(lián)合AUY922能夠成功抑制并逆轉(zhuǎn)氟維司群帶來的Erb B家族蛋白反饋性激活效應(yīng),進(jìn)而抑制下游增殖相關(guān)PI3K/AKT和ERK信號(hào)通路。4.利用MTT實(shí)驗(yàn)、平板克隆實(shí)驗(yàn)檢測(cè)雙藥聯(lián)合對(duì)于細(xì)胞生長(zhǎng)的抑制作用,利用流式細(xì)胞技術(shù)檢測(cè)雙藥聯(lián)合作用對(duì)于細(xì)胞周期的阻滯。5.人源腫瘤組織的離體驗(yàn)證實(shí)驗(yàn)中用western blotting技術(shù)進(jìn)一步證實(shí)了AUY922對(duì)于氟維司群的反饋激活效應(yīng)的逆轉(zhuǎn)作用。研究結(jié)果:1.氟維司群能夠在48小時(shí)內(nèi)降低MCF-7和T47D細(xì)胞中的ER的水平。2.氟維司群在降低MCF-7和T47D細(xì)胞ER水平的同時(shí)能反饋性激活Erb B家族蛋白,同時(shí)p-EGFR、p-HER2、p-HER3及p-HER4的表達(dá)量增加,表明其功能活化。這一反饋性激活作用在氟維司群作用的短時(shí)間(48小時(shí))內(nèi)引起的。3.AUY922單藥能夠有效抑制MCF-7和T47D中的HSP90活性,進(jìn)一步檢測(cè)發(fā)現(xiàn)氟維司群對(duì)于HSP90活性并無影響。AUY922能夠有效抑制PI3K/AKT和ERK信號(hào)通路的活性。4.在氟維司群作用24小時(shí)之后的基礎(chǔ)上,進(jìn)一步聯(lián)合AUY922能夠成功抑制并逆轉(zhuǎn)氟維司群引起的Erb B家族蛋白的反饋性激活效應(yīng),并且有效抑制了下游增殖相關(guān)PI3K/AKT和ERK信號(hào)通路的活性。5.在細(xì)胞功能學(xué)實(shí)驗(yàn)中,雙藥聯(lián)合能夠有效抑制細(xì)胞生長(zhǎng),在平板克隆形成實(shí)驗(yàn)中發(fā)現(xiàn),雙藥聯(lián)合能夠更加有效抑制細(xì)胞克隆形成。細(xì)胞周期檢測(cè)中發(fā)現(xiàn),在ER+乳腺癌細(xì)胞中雙藥聯(lián)合能夠更有效的阻滯細(xì)胞周期。6.人源腫瘤組織的體外驗(yàn)證實(shí)驗(yàn)證明,氟維司群的治療確實(shí)能夠反饋性激活Erb B家族蛋白,引起下游PI3K/AKT和ERK信號(hào)通路的活化,聯(lián)合應(yīng)用AUY922能夠成功逆轉(zhuǎn)這種反饋性激活效應(yīng)。進(jìn)一步證實(shí)了細(xì)胞系中的實(shí)驗(yàn)結(jié)論。研究結(jié)論:本研究發(fā)現(xiàn)并證實(shí)了Erb B受體家族及其下游增殖相關(guān)通路PI3K/AKT和ERK信號(hào)通路在氟維司群治療后能夠發(fā)生反饋性激活,而HSP90抑制劑AUY922能夠多靶點(diǎn)抑制這些反饋性活化的蛋白活性,同時(shí),這一實(shí)驗(yàn)結(jié)果在乳腺癌細(xì)胞系及新鮮乳腺癌離體標(biāo)本中均得到了證實(shí)。
[Abstract]:Background and objective: endocrine therapy as adjuvant therapy for breast cancer treatment strategies that can significantly improve the prognosis of [1] patients with hormone receptor positive breast cancer, fulvestrant (Fulvestrant) is a group of common endocrine drugs [2]. protein analysis found that long-term estrogen deprived cells (long-term estrogen deprivation cells, LTED cells) the enhanced Erb B family of receptor mediated PI3K/m TOR signaling pathway activity. There is evidence that the activation of [3-7] PI3K/AKT signaling pathway in estrogen treatment of drug-resistant breast cancer patients, and thus play an important role in cell growth and proliferation. However, the results from the current study, the estrogen resistant cells, alone the application of EGFR inhibitor gefitinib and activation of signal regulatory networks [8]. current related research shows that AKT feedback cannot inhibit tumor cell growth and proliferation resistance The process is very complicated, simple interference or inhibition of a specific target for the inhibition of cell proliferation to kill cancer cells may be very limited. Therefore, if can also inhibit tumor cell growth in a number of key proteins for inhibiting the growth of tumor.HSP90 expression seems to be more effective broad-spectrum intracellular chaperone protein folding. Proteins related to many malignant phenotype of tumor cells are dependent on HSP90 to exert its biological effects. The related clinical data showed that the expression of HSP90 in tumor tissue compared with adjacent normal tissue was 2-3 times higher, but with a poor clinical outcome was significantly related to the [9,10]. study found that high expression and poor prognosis of breast cancer HSP90 chaperone the effect of.HSP90 is closely related to, and independent factors of prognosis of breast cancer with high expression of HER2 positive, ER positive, larger tumor size, histology High grade and lymph node metastasis of [11]. has significant correlation showed that HSP90 can enhance the breast cancer cells for resistance to hormone therapy, and drug resistance related [12].AUY922 hormone therapy is the second generation of HSP90 inhibitors, which can compete with HSP90 in ATP binding sites, thereby inhibiting the biological activity of HSP90, resulting in many of the key protein in cells can not be folded to lose normal biological function. Due to the feedback activation effect of fulvestrant is an important cause of breast cancer cells and drug resistance, multiple pathways and multiple pathway related proteins involved in this process, however, there is still no about whether AUY922 can reverse the feedback activation effect of related research. We speculate that AUY922 may be able to inhibit the activity of HSP90, thereby inhibiting the Erb B family and PI3K/AKT and ERK signaling pathway, the reversal of fluoride The activation effects brought by our group dimension feedback, provide relevant clinical trials support new theory basis for combination therapy. Methods: 1. using Western blotting technique to detect effects of fulvestrant monotherapy for MCF-7 and T47D cells at the ER level, and within a short period of time for the Erb feedback activation of B family proteins further, the activation of PI3K/AKT and downstream of the ERK pathway. And RT-PCR is used to detect the changes of the.2. Erb B family m RNA level in fulvestrant treatment after the detection of AUY922 single drug for the detection of PI3K/AKT and ERK signal pathway of the inhibitory effect of.3.Western Blotting Technology Based on fulvestrant treatment combined with AUY922 can inhibit and reverse fulvestrant brought Erb B protein family feedback activation effect using Western Blotting Technology, and then inhibit the downstream proliferation related PI3K/AKT signal pathways of ERK and.4. Experiments using MTT assay colony, the two drug combination for inhibition of cell growth, tissue block.5. for human tumor cell cycle by flow cytometry double medicine combined effect of in vitro experiments using Western blotting technology further confirmed that AUY922 for fulvestrant feedback activation effect of reversal effect results: 1.. Fulvestrant can reduce MCF-7 and T47D cells in ER within 48 hours of the level of.2. of fulvestrant in MCF-7 and T47D cells and reduce the level of ER can feedback activation of Erb B protein family, and p-EGFR, p-HER2, expression of p-HER3 and p-HER4 increase, show that its functions activation. This feedback activation of fulvestrant in short time (48 hours) the role of.3.AUY922 in the single drug can effectively inhibit MCF-7 and T47D activity in HSP90, further detection of fulvestrant For does not affect the activity of HSP90.AUY922 can effectively inhibit PI3K/AKT and ERK pathway in.4. based fulvestrant 24 hours later, further combined with AUY922 can successfully inhibit and reverse fulvestrant feedback activation effect induced by Erb B protein family, and can inhibit the activation of.5. and downstream proliferation related PI3K/AKT and the ERK signaling pathway in cell function experiments, two drug combination can effectively inhibit the cell growth and colony formation in the two drug combination can effectively inhibit the formation of cell clones. Found that cell cycle detection, in ER+ breast cancer cells by two drug combination can prove the in vitro experiments of cell cycle arrest.6. in human tumor tissue more efficiently, the treatment of fulvestrant can feedback activation of Erb B protein family, caused by the downstream PI3K/AKT and ERK signaling pathway The activation of the combined application of AUY922 can successfully reverse the feedback activation effect. Experimental results further confirmed the cell line. Conclusion: This study found and confirmed the Erb B receptor and its downstream pathways related to PI3K/AKT proliferation and ERK signaling pathways occur feedback activation in the treatment of fulvestrant can, HSP90 inhibitor AUY922 can these feedback target inhibition activated protein activity, at the same time, this results in breast cancer cell lines and fresh breast cancer specimens were confirmed.

【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R737.9

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