發(fā)布時間：2018-03-03 10:19

  本文選題：前列腺癌　切入點：自噬　出處：《第四軍醫(yī)大學》2014年碩士論文　論文類型：學位論文

【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人口進入老齡化，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胞的特異性，對正常的身體組織也會產(chǎn)生嚴重的破壞，在臨床應用上受到一定的限制，克服化療藥物的副作用，提高殺滅癌細胞的特異性，是目前比較重要的研究課題之一。 激素非依賴性前列腺癌自噬作用缺失、抗凋亡能力增強可能是其腫瘤細胞增殖的原因。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可以促進細胞凋亡，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，特異地增強前列腺癌細胞的自噬和凋亡，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引物設計軟件設計含有BamHⅠ酶切位點的SA的特異性引物。以Core SA作為模板，利用PCR技術(shù)擴增出SA，長度為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序。SA-beclin1測序結(jié)果與預期完全一致，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原核表達菌，用IPTG誘導表達融合蛋白SA-beclin1，通過優(yōu)化表達條件，選擇37℃，IPTG濃度0.5mmol/L，誘導5h。用鎳親和凝膠層析柱純化融合蛋白，Western blot鑒定融合蛋白表達情況。結(jié)果顯示IPTG誘導后SA-beclin1融合蛋白(含His標簽)在大腸桿菌中高效表達。SDS-PAGE分析表達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鑒定其相對分子量約為72000，與預期相符。包涵體經(jīng)變性復性后，亦可得到與目的條帶大小相符的蛋白。 設計并合成小肽NuBCP-9，并分別標記生物素和FITC，得到純度90%的生物素標記小肽NuBCP-9，為后續(xù)實驗提供基礎。 以上結(jié)果表明，成功構(gòu)建了重組質(zhì)粒并表達純化了SA-beclin1融合蛋白，得到生物素標記的小肽NuBCP-9，為進一步研究SA-beclin1的功能及臨床應用奠定了基礎。
[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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