用慢病毒PRMT1侵染肺癌细胞A549得稳转细胞及其功能的鉴定
时间:2019-04-13 14:23 来源:毕业论文 作者:毕业论文 点击:次
| 概要：目的：现在人们“谈癌色变”，而肺癌是我们生活中常见的一种癌症。据统计，肺癌的发病率和死亡率每年呈上升趋势，世界每一年得病率在90万左右。我国近10年内恶性肿瘤死亡率平均年递增以肺癌最快。因此，对肺癌细胞相关基因的研究具有重要意义，能对预防和治疗肺癌提供基础医学理论依据。本文通过进行PRMT1测序，病毒克隆构建，病毒包装，病毒载体感染肺癌细胞等一系列工作，得到PRMT1基因敲降的稳转细胞，并进行稳转细胞功能的鉴定。方法:用PRMT1-shRNA（含绿色荧光）病毒来敲降肺癌细胞A549的PRMT1，用荧光显微镜检测绿色荧光蛋白，用嘌呤霉素药筛得重组质粒，western blot做PRMT1蛋白表达的鉴定及其功能简单分析。结果：得到PRMT1基因敲降的肺癌A549稳转细胞。34457 毕业论文关键词：肺癌；PRMT1；慢病毒；A549细胞。 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. Keywords: Lung cancer; PRMT1; Lentivirus; A549 cells. 目录 引言 1 1.材料与方法 3 1.1仪器与材料 3 1.1.1材料 3 1.1.2仪器 3 1.1.3试剂 4 1.2方法 6 1.2.1.基因的获得 6 1.2.2回收 6 1.2.3连接 6 1.2.4转化 6 1.2.5碱裂解法抽取质粒 6 1.2.6重组质粒克隆的鉴定 6 1.2.7质粒DNA的含量及纯度鉴定 6 1.2.8细胞转染 6 1.2.9病毒的收获及浓缩 6 1.2.10 慢病毒滴度测定 7 1.2.11 慢病毒感染目的细胞 7 1.2.12感染后的细胞检测方法 8 1.2.13加嘌呤霉素进行药筛 8 1.2.14蛋白提取及Western Blot检测 8 2.结果与分析 11 2.1 慢病毒的包装结果 11 2.2免疫荧光实验 12 2.3嘌呤霉素药筛实验 12 2.4 Western Bolt检测A549-lentivirus -PRMT1蛋白质表达水平 13 3.讨论 13 4.致谢 15 参考文献 15 引言： 1.肺癌的现状 (责任编辑：qin) |