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用慢病毒PRMT1侵染肺癌細胞A549得穩轉細胞及其功能的鑒定

時間:2019-04-13 14:23來源:畢業論文
用PRMT1-shRNA(含綠色熒光)病毒來敲降肺癌細胞A549的PRMT1,用熒光顯微鏡檢測綠色熒光蛋白,用嘌呤霉素藥篩得重組質粒,western blot做PRMT1蛋白表達的鑒定及其功能簡單分析

概要:目的:現在人們“談癌色變”,而肺癌是我們生活中常見的一種癌癥。據統計,肺癌的發病率和死亡率每年呈上升趨勢,世界每一年得病率在90萬左右。我國近10年內惡性腫瘤死亡率平均年遞增以肺癌最快。因此,對肺癌細胞相關基因的研究具有重要意義,能對預防和治療肺癌提供基礎醫學理論依據。本文通過進行PRMT1測序,病毒克隆構建,病毒包裝,病毒載體感染肺癌細胞等一系列工作,得到PRMT1基因敲降的穩轉細胞,并進行穩轉細胞功能的鑒定。方法:用PRMT1-shRNA(含綠色熒光)病毒來敲降肺癌細胞A549的PRMT1,用熒光顯微鏡檢測綠色熒光蛋白,用嘌呤霉素藥篩得重組質粒,western blot做PRMT1蛋白表達的鑒定及其功能簡單分析。結果:得到PRMT1基因敲降的肺癌A549穩轉細胞。34457
畢業論文關鍵詞:肺癌;PRMT1;慢病毒;A549細胞。
Acquisition of stably transfected cells using PRMT1 -lentivirus infection of lung cancer A549 cells and identification of their functions
Abstract: Target: nowadays, most people will turn pale at the mentioning of cancers, and we all know that lung cancer is a common type of cancer in our lives.According to some information statistics,the morbidity and mortality of lung cancer is on the rise year by year whose number is approximately 900000 every year. In China, the fastest average annual increasing rate of the mortality of a malignant tumor is lung cancer in recent 10 years. Therefore, the study about related genes in lung cancer cells is of great significance, to provide basic medical theory basis for the prevention and treatment of lung cancer. This article mainly studied about the acquisition cells that PRMT1 knockdown and the identification of stably infected cells by the lentivirus through the sequencing of PRMT1, virus cloned construction , virus packaging,the transfection of viral vector to lung cancer cells and so on a series of work. Methods: use PRMT1 - shRNA (containing green fluorescence) virus to knock down the gene PRMT1 in A549 lung cancer cells;use a fluorescence microscopegreen to test the fluorescent protein;use puromycin to screen recombinant plasmid and through western bloting to analysis the expression of PRMT1 proteins in the infected lung cancer cells. Results:having got the lung cancer cells A549 infected by PRMT1-shRNA virus. 源¥自%六^^維*論-文+網=www.aftnzs.live
Keywords: Lung cancer; PRMT1; Lentivirus; A549 cells.
目錄
引言    1
1.材料與方法    3
     1.1儀器與材料    3
1.1.1材料    3
1.1.2儀器    3
1.1.3試劑    4
     1.2方法    6
1.2.1.基因的獲得    6
1.2.2回收    6
1.2.3連接    6
1.2.4轉化    6
1.2.5堿裂解法抽取質粒    6
1.2.6重組質粒克隆的鑒定    6
1.2.7質粒DNA的含量及純度鑒定    6
1.2.8細胞轉染    6
1.2.9病毒的收獲及濃縮    6
1.2.10 慢病毒滴度測定    7
1.2.11 慢病毒感染目的細胞     7
1.2.12感染后的細胞檢測方法    8
1.2.13加嘌呤霉素進行藥篩    8
1.2.14蛋白提取及Western Blot檢測    8
2.結果與分析    11
2.1 慢病毒的包裝結果    11
2.2免疫熒光實驗    12
2.3嘌呤霉素藥篩實驗    12
2.4 Western Bolt檢測A549-lentivirus -PRMT1蛋白質表達水平    13
3.討論    13
4.致謝    15
參考文獻    15
引言:
1.肺癌的現狀 用慢病毒PRMT1侵染肺癌細胞A549得穩轉細胞及其功能的鑒定:http://www.aftnzs.live/shengwu/20190413/31963.html
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