三种方法转染Math1基因效率和细胞毒性研究

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体外携带目的 基因Math1对HEK293T细胞的转染效率及细胞毒性的大小,以期筛选理想的基因转染载体材料.方法 选用重组腺病毒、LipofectamineTM 2000、Superfect纳米材料作为转染载体,携带含有增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)报告基因的Math1-EGFP基因及真核表达质粒pRK5-Math1-EGFP,并且根据不同转染载体说明步骤优化体外转染条件,分别转染293T细胞.在一定的时间内利用荧光显微镜观察细胞转染结果并计数阳性细胞,采用MTT(噻唑蓝)法检测三种不同载体体外细胞毒性,应用RT-PCR(逆转录聚合酶链反应)技术检测转染阳性细胞中目的 基因Math1的mRNA表达情况.结果 在优化的体外转染条件下,重组腺病毒载体介导的细胞转染率达到了94％以上,脂质体和商品化Supeffect纳米材料转染的细胞中,荧光蛋白表达率分别为73％和80％以上,同时,商品化Superfect纳米材料在达到80％转染率的条件下,细胞存活率为90％.应用RT-PCR方法 证实,三种载体转染细胞均有Math1基因的mRNA的表达.结论 商品化Supeffect纳米材料作为一种新型的、安全有效的纳米转染材料,能够成功携带耳聋治疗关键基因Math1转染293T细胞,以期在内耳基因治疗的研究当中得到有效的应用.%Objective To determine most effective gene transfection vectors by comparing their transfection efficiency and cytotoxicity to 293T cells. Methods Recombinant adenovirus, lipofectamine 2000 and Superfect nanoparticles were used as gene vectors carrying the Mathl-EGFP gene and pRK5-Mathl-EGFP plasmid, which report gene enhanced green fluorescent protein (EGFP). Expression positive cells were counted under a fluorescence microscope and the survival rate of 293T cells calculated using MTT assay. RT-PCR was used to determine integration of mRNA expression in positive 293T cells. Results By optimizing transfection conditions, the highest transfection efficiency was above 94% for recombinant adenovirus, 73% for Lipofectamine 2000 and 80% for Superfect nanoparticles. The cellular viability was about 90% for Superfect nanoparticles. Conclusion A high transfection efficiency and low cytotoxicity of cells was achieved with Superfect-pRK5- Mathl-EGFP complex nanoparticles, which may be used as a safe and effective vector in research of inner ear diseases.