本文選題：前列腺癌　切入點(diǎn)：自噬　出處：《第四軍醫(yī)大學(xué)》2014年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：前列腺癌（prostate cancer，PCa）是一種老年男性泌尿系統(tǒng)的疾病，隨著生活水平和診療手段的不斷提高以及我國(guó)人口進(jìn)入老齡化，前列腺癌發(fā)病率逐年增高。但是目前的醫(yī)療水平，使前列腺癌確診時(shí)基本已是中晚期，而且傳統(tǒng)的治療手段如手術(shù)治療、激素治療等療效欠佳，放化療等治療方法由于缺乏靶細(xì)胞的特異性，對(duì)正常的身體組織也會(huì)產(chǎn)生嚴(yán)重的破壞，在臨床應(yīng)用上受到一定的限制，克服化療藥物的副作用，提高殺滅癌細(xì)胞的特異性，是目前比較重要的研究課題之一。 激素非依賴(lài)性前列腺癌自噬作用缺失、抗凋亡能力增強(qiáng)可能是其腫瘤細(xì)胞增殖的原因。自噬相關(guān)基因beclin1是目前新發(fā)現(xiàn)的抑癌基因，源于核孤兒受體的小肽NuBCP-9可以促進(jìn)細(xì)胞凋亡，前列腺特異性膜抗原(prostate-specific membraneantigen，PSMA)的適配子能特異地與前列腺癌細(xì)胞結(jié)合。本課題擬利用鏈霉親和素-生物素技術(shù)將beclin1與小肽NuBCP-9以及PSMA適配子偶聯(lián)在一起，特異地增強(qiáng)前列腺癌細(xì)胞的自噬和凋亡，試圖為前列腺癌的治療尋找一條有效的新途徑。 1、構(gòu)建SA-beclin1原核表達(dá)載體 從GenBank中檢索出核心鏈霉親和素Core SA基因的DNA序列后，，利用in-Fusion引物設(shè)計(jì)軟件設(shè)計(jì)含有BamHⅠ酶切位點(diǎn)的SA的特異性引物。以Core SA作為模板，利用PCR技術(shù)擴(kuò)增出SA，長(zhǎng)度為381bp。自噬相關(guān)基因beclin1已在前期工作中構(gòu)建入原核表達(dá)載體pQE80L，用in-Fusion方法連接PCR產(chǎn)物SA和pQE80L-beclin1，酶切鑒定并送公司測(cè)序。SA-beclin1測(cè)序結(jié)果與預(yù)期完全一致，重組載體構(gòu)建成功，命名為pQE80L-SA-beclin1。2、融合蛋白SA-beclin1的原核表達(dá)與純化 將重組載體pQE80-SA-beclin1轉(zhuǎn)化入大腸桿菌Rosetta-gami原核表達(dá)菌，用IPTG誘導(dǎo)表達(dá)融合蛋白SA-beclin1，通過(guò)優(yōu)化表達(dá)條件，選擇37℃，IPTG濃度0.5mmol/L，誘導(dǎo)5h。用鎳親和凝膠層析柱純化融合蛋白，Western blot鑒定融合蛋白表達(dá)情況。結(jié)果顯示IPTG誘導(dǎo)后SA-beclin1融合蛋白(含His標(biāo)簽)在大腸桿菌中高效表達(dá)。SDS-PAGE分析表達(dá)的融合蛋白以包涵體為主，經(jīng)鎳親和凝膠層析柱純化得到融合蛋白，Western blot鑒定其相對(duì)分子量約為72000，與預(yù)期相符。包涵體經(jīng)變性復(fù)性后，亦可得到與目的條帶大小相符的蛋白。 設(shè)計(jì)并合成小肽NuBCP-9，并分別標(biāo)記生物素和FITC，得到純度90%的生物素標(biāo)記小肽NuBCP-9，為后續(xù)實(shí)驗(yàn)提供基礎(chǔ)。 以上結(jié)果表明，成功構(gòu)建了重組質(zhì)粒并表達(dá)純化了SA-beclin1融合蛋白，得到生物素標(biāo)記的小肽NuBCP-9，為進(jìn)一步研究SA-beclin1的功能及臨床應(yīng)用奠定了基礎(chǔ)。
[Abstract]:Prostate cancer (PCA) is a disease of the urinary system in elderly men. With the improvement of living standard and diagnosis and treatment means and the aging of population in our country, the incidence of prostate cancer is increasing year by year. The diagnosis of prostate cancer is basically late, and the traditional treatment methods, such as surgery, hormone therapy and so on, are not good. The radiotherapy and chemotherapy methods lack the specificity of target cells. It is one of the most important research topics to overcome the side effects of chemotherapeutic drugs and to improve the specificity of killing cancer cells. The loss of autophagy and the increase of anti-apoptotic ability of steroid independent prostate cancer may be the reasons for the proliferation of tumor cells. Autophagy associated gene beclin1 is a newly discovered tumor suppressor gene. Small peptide NuBCP-9, derived from nuclear orphan receptors, promotes apoptosis. The aptamers of prostate-specific membrane antigens (PSMA) can specifically bind to prostate cancer cells. In this study, beclin1 was coupled with small peptide NuBCP-9 and PSMA aptamers by streptavidin-biotin technique. It specifically enhances autophagy and apoptosis of prostate cancer cells, and attempts to find a new effective way for the treatment of prostate cancer. 1. Construction of prokaryotic expression vector of SA-beclin1. The DNA sequence of core streptavidin Core SA gene was retrieved from GenBank. The specific primers of SA containing BamH 鈪

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