摘要
背景:基因载体是基因治疗中重要的影响因素,阳离子脂质体细胞毒性低,转染效率高,是一种很有前景的基因转染载体。目的:评价两种阳离子脂质体介导基因转染宫颈癌细胞的转染效果,优化它们对宫颈癌细胞的转染条件。设计、时间及地点:以Hela细胞为观察对象,分组设计。实验于2008-03/06在大连民族学院生命科学学院国家民委-教育部重点实验室完成。材料:质粒pGFP-N2购自Clontech公司,Hela细胞由大连民族学院的实验室保存。方法:首先采用DNA延滞实验考察阳离子脂质体与DNA的结合能力,然后用进行细胞转染实验研究脂质体的转染效率,最后通过四甲基偶氮唑盐法考察脂质体对细胞的毒性。主要观察指标:采用DNA延滞实验观察阳离子脂质体Lipofectamine2000和DOTAP与DNA的结合能力,以报告基因(绿色荧光蛋白基因pGFP-N2),分析阳离子脂质体Lipofectamine2000和DOTAP转染Hela细胞转染效率和细胞毒性。结果:Lipofectamine2000和DOTAP与DNA均有很强的结合力;以绿色荧光蛋白基因为报告基因,Lipofectamine2000转染率较高;当Lipofectamine2000与DNA质量比为3∶1时,转染效率最高,约72%,是DOTAP转染细胞最高活性的2.5倍;在最佳转染剂量时,2种试剂的细胞存活率均在75%以上,Lipofectamine2000的毒性相对较小。结论:对Hela细胞而言,Lipofectamine2000较DOTAP具有更高的转染效率和较低的细胞毒性。
BACKGROUND： Gene vector is an important factor in gene therapy. Cationic liposome is a promising vector in gene transfection for its low cytotoxicity and high transfection efficiency.
OBJECTIVE： To evaluate the gene transfection efficiency of two types of cationic liposomes to cervical carcinoma cell, in addition to optimize the transfection conditions.
DESIGN, TIME AND SETTING： The grouping design based on Hela ceils was performed in the Key Laboratory of Biotechnology and Bioresources Utilization in Life Science College, Dalian Nationalities Unviersity from March to June in 2008.
MATERIALS. Plasmid pGFP-N2 was purchased from Clontech Company. Hela cells were provided by Cell Culture Laboratory in Life Science College, Dalian Nationalities University.
METHODSDNA arrearage assay was firstly applied to determine the capability of cationic liposomes with DNA. Then the transfection efficiency and the cytotoxicity of liposomes were examined using methyl thiazolyl tetrazolium method.
MAIN OUTCOME MEASURES： The binding capacity of Lipofectamine 2000 and DOTAP was detected by DNA arrearage assay. The transfection efficiency and the cytotoxicity of Hela cell transfected with Lipofectamine 2000 and DOTAP were analyzed by reporter gene （green fluorescent protein gene pGFP-N2）, RESULTS： Lipofectamine 2000 and DOTAP had strong binding capacity with DNA; Lipofectamine 2000 could get higher transfection efficiency when GFP served as reporter gene; when the weight ratio of Lipofectamine 2000 and DNA was 3：1, the highest transfection efficiency 72%, which was 2.5 folds of the highest efficiency of DOTAP was obtained. On the other hand, the cytotoxicity assay showed that over 75% cell viability could be achieved both by Lipofectamine 2000 and DOTAP on the optimal transfection condition, and Lipofectamine 2000 had relative lower cytotoxicity.
CONCLUSION： Lipofectamine 2000 has relatively higher transfection rate and lower cytotoxicity than DOTAP to Hela cell.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2009年第11期2119-2122,共4页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
国家自然科学基金(20876027)
辽宁省科技厅博士启动基金资助项目(20031082)
辽宁省教育厅高等学校科学研究项目(20040084)~~
