本文選題：乳腺癌　切入點(diǎn)：乳腺癌轉(zhuǎn)移　出處：《華東師范大學(xué)》2015年博士論文　論文類型：學(xué)位論文

【摘要】：乳腺癌是女性最常見(jiàn)腫瘤。在過(guò)去的幾十年間,隨著早診斷的意識(shí)和治療手段的提高,乳腺癌患者的死亡率有了一定程度的降低。但是,每年仍有數(shù)以萬(wàn)計(jì)的患者死于乳腺癌。對(duì)于乳腺癌患者來(lái)說(shuō),最差的預(yù)后是發(fā)生了腫瘤轉(zhuǎn)移,因?yàn)榻^大部分的乳腺癌患者直接或間接地死于乳腺癌轉(zhuǎn)移。傳統(tǒng)的乳腺癌療法包括手術(shù)治療,放射療法,以及系統(tǒng)療法。手術(shù)療法聯(lián)合放療能清除絕大部分的原位乳腺腫瘤。手術(shù)治療前或后對(duì)患者施以系統(tǒng)療法(包括化療,激素療法和分子靶向療法)在清除一些殘余的原位腫瘤細(xì)胞的同時(shí)也能縮小轉(zhuǎn)移灶的大小。然而,這些治療方法其目的均是針對(duì)腫瘤細(xì)胞的生存。因此,研發(fā)靶向腫瘤轉(zhuǎn)移的新型藥物能為臨床治療乳腺癌提供新的治療策略并且能降低乳腺癌轉(zhuǎn)移的風(fēng)險(xiǎn)。1999年,Davis及其同事發(fā)現(xiàn)了一個(gè)調(diào)控肌肉萎縮癥的ferlin家族的新成員。他們將這個(gè)蛋白命名為Myoferlin (MYOF)。近年來(lái),人們也逐漸意識(shí)到MYOF在乳腺癌中的重要作用。已有研究人員證明MYOF在臨床的浸潤(rùn)性乳腺癌患者的樣本中有異常高的表達(dá);此外,還有證據(jù)表明MYOF在乳腺癌的浸潤(rùn)和上皮-間質(zhì)轉(zhuǎn)化(EMT)過(guò)程中起重要作用,提示MYOF是一個(gè)乳腺癌轉(zhuǎn)移的相關(guān)蛋白且可以作為治療乳腺癌轉(zhuǎn)移的一個(gè)潛在靶標(biāo)�；谏鲜霰尘�,我們對(duì)本實(shí)驗(yàn)室的小分子化合物庫(kù)進(jìn)行了篩選。首先,我們利用體外乳腺癌轉(zhuǎn)移相關(guān)實(shí)驗(yàn)篩選出11個(gè)具有較強(qiáng)體外抗乳腺癌轉(zhuǎn)移活性的小分子。利用Matrigel侵襲實(shí)驗(yàn)我們對(duì)這11個(gè)小分子進(jìn)行了進(jìn)一步的篩選。我們從中得到了一個(gè)新型的具最強(qiáng)抗乳腺癌轉(zhuǎn)移活性的小分子WJ460。隨后,我們利用小鼠乳腺癌自發(fā)性轉(zhuǎn)移模型檢測(cè)了WJ460是否也具有較強(qiáng)的體內(nèi)抗乳腺癌轉(zhuǎn)移的能力。實(shí)驗(yàn)結(jié)果表明,WJ460能顯著抑制乳腺癌細(xì)胞向小鼠各遠(yuǎn)端器官的轉(zhuǎn)移。為了探究WJ460抑制乳腺癌轉(zhuǎn)移的具體分子機(jī)制,我們借助了生物素分子探針。通過(guò)將生物素偶聯(lián)到小分子WJ460上,我們成功“釣取”了WJ460的直接作用靶蛋白。通過(guò)質(zhì)譜分析,我們發(fā)現(xiàn)并鑒定該蛋白是MYOF。此外,我們通過(guò)親和pull-down實(shí)驗(yàn)對(duì)這一結(jié)果進(jìn)行了驗(yàn)證。于此同時(shí),免疫熒光的結(jié)果也顯示W(wǎng)J460和MYOF確實(shí)存在共定位。為了尋找MYOF和WJ460結(jié)合的具體部位,我們構(gòu)建了不同的MYOF缺失體。通過(guò)親和pull-down實(shí)驗(yàn)我們發(fā)現(xiàn)只有含有C2D結(jié)構(gòu)域的MYOF缺失體能與WJ460相結(jié)合。接下來(lái),我們探究了WJ460是否影響MYOF所調(diào)節(jié)的與乳腺癌轉(zhuǎn)移相關(guān)的生物學(xué)過(guò)程。實(shí)驗(yàn)結(jié)果表明,WJ460能明顯抑制MYOF的靶基因基質(zhì)金屬蛋白酶1(MMP1)的基因及蛋白表達(dá)。通過(guò)3D培養(yǎng),免疫熒光,實(shí)時(shí)定量PCR及蛋白免疫印跡實(shí)驗(yàn),我們發(fā)現(xiàn)WJ460能逆轉(zhuǎn)乳腺癌的上皮-間質(zhì)轉(zhuǎn)化(EMT)過(guò)程從而降低乳腺癌轉(zhuǎn)移能力。此外,我們利用人源的受體酪氨酸激酶芯片檢測(cè)了WJ460對(duì)受體酪氨酸激酶活性(磷酸化形式)的影響。實(shí)驗(yàn)結(jié)果表明,多個(gè)受體酪氨酸激酶活性均能被WJ460所抑制。最后,通過(guò)小鼠實(shí)驗(yàn)性肺轉(zhuǎn)移模型,我們發(fā)現(xiàn)knock-down MYOF和WJ460處理有類似的抑制乳腺癌轉(zhuǎn)移的效果。同時(shí),我們的結(jié)果還顯示,knock-down MYOF和WJ460處理均能抑制乳腺癌肺轉(zhuǎn)移的外滲與惡性增殖。綜上所述,我們的研究表明WJ460可作為第一個(gè)靶向MYOF的先導(dǎo)化合物,同時(shí)為WJ460治療乳腺癌轉(zhuǎn)移提供了一定的理論依據(jù)。
[Abstract]:Breast cancer is the most common female cancer. In the past few decades, with the awareness and treatment of early diagnosis of breast cancer mortality has decreased to a certain degree. However, there are still tens of thousands of patients die of breast cancer each year. For patients with breast cancer, the worst prognosis is the occurrence of the tumor metastasis that is because most of the breast cancer patients directly or indirectly died of breast cancer metastasis. Breast cancer therapy including traditional surgery, radiotherapy, and systemic therapy. Surgical therapy combined with radiotherapy can remove most of the in situ mammary gland tumor. Surgical treatment before or after therapy to patients of system (including chemotherapy, hormone therapy and molecular targeted therapy) can also reduce the size of the metastases in the removal of some in situ residual tumor cells at the same time. However, these treatments the aim is for students of tumor cells Deposit. Therefore, development of new drugs targeting tumor metastasis can provide new therapeutic strategies for the treatment of breast cancer and can reduce the risk of breast cancer metastasis in.1999, Davis and colleagues found a new member of the ferlin family regulate muscle atrophy. They will be the protein named Myoferlin (MYOF) in recent years. Also, people gradually realized the important role of MYOF in breast cancer. Researchers have demonstrated that MYOF in the clinical expression of abnormally high of breast cancer patients in the sample; in addition, there is evidence that MYOF in breast cancer invasion and epithelial mesenchymal transition (EMT) plays an important role in the process, prompt MYOF is a potential target for a breast cancer metastasis related protein and can be used as a treatment for breast cancer metastasis. Based on the above background, we in the laboratory of small molecular libraries were screened. First of all, we benefit The selected 11 has strong anti breast cancer activity in vitro transfer of small molecules in vitro and metastasis of breast cancer. The use of Matrigel invasion experiment we consider these 11 small molecules were further screened. We get a new type of small molecules with the strongest activity against breast cancer metastasis WJ460. then, we use the mice breast cancer model with spontaneous metastasis detected whether WJ460 also has anti breast cancer metastasis ability in vivo. The experimental results show that WJ460 can significantly inhibit the metastasis of breast cancer cells into mice of the remote organs. In order to explore the molecular mechanism of WJ460 inhibiting the metastasis of breast cancer, we used the biotin molecules through biological probe. By coupling the small molecule WJ460, we successfully obtained the direct effect of WJ460 target protein. By mass spectrum analysis, we found and identified the protein is MYOF. addition ,鎴戜滑閫氳繃浜插拰pull-down瀹為獙瀵硅繖涓，

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