摘要
目的比较腺病毒、脂质体、纳米材料三种不同类型的载体携带目的基因对293T细胞的转染效率及细胞毒性的大小,筛选理想的基因载体。方法用重组腺病毒、LipofectamineTM 2000、EntransterTM-D纳米材料为转染载体,携带含有增强型绿色荧光蛋白(EGFP)报告基因的Math1-EGFP基因及真核表达质粒pRK5-Math1-EGFP,按照转染试剂盒说明优化其转染条件,分别转染293T细胞。24小时后在共聚焦显微镜下观察转染结果并计数阳性细胞率,采用RT-PCR法检测转染细胞中Math1基因mRNA的表达,用MTT法检测不同转染条件下,不同转染载体对293T细胞的细胞毒性。结果经优化转染条件,重组腺病毒载体转导的细胞中荧光蛋白表达几乎为95%以上,LipofectamineTM 2000、EntransterTM-D纳米材料载体转染的细胞中荧光蛋白表达也分别达到70%和80%以上,而EntransterTM-D纳米材料载体在最高转染效率的剂量下仍然保持80%以上的细胞生存率。RT-PCR进一步证实,三种载体转染细胞均有Math1基因mRNA的表达。结论EntransterTM-D纳米载体作为一种新型纳米聚合物转染试剂,能成功携带内耳基因治疗关键基因Math1基因转染293T细胞,并表现了低毒、高效性能。
Objective To get ideal gene vectors trough investigating the transfection efficiency and cytotoxicity to 293T cells for three different reagents. Methods Recombinant adenovirus, Lipofectamine^TM2000 and Entranster^TM-D nanoparticle were used as different transfection vectors with Math1-EGFP gene and pRKS-Math1-EGFP plasmid, which carrying report gene enhanced green fluorescent protein （EGFP）. The positive ceils were counted by eonfocal microscopy and the cellular viability was calculated by MTT assay in different conditions. RT-PCR was used to determine the integration of mRNA expression in transfected 293T cells. Results By optimizing the transfection conditions, the highest transfection efficiency of recombinant adenovirus was above 95%, and LipofectamineTM2000 as well as Entranster^TM-D nanoparticle was more than70% and 80% respectively. The cellular viability was about 80% for EntransterW-D nanopartiele vectors. Conclusion A high transfection efficiency and low cytotoxicity of 293T cells is achieved with Entranster^TM-DpRK5- Math1-EGFP complex nanoparticle which indicate is a safe and effective vector .
出处
《中华耳科学杂志》
CSCD
2009年第4期347-351,共5页
Chinese Journal of Otology
基金
国家自然科学基金(面上项目30871398
30571017
30000189)
海外青年合作基金(30628030)
北京自然科学基金(7042061)
国家863计划(2007AA02Z150)
国家自然科学基金重点项目(30730040)资助
