Abstract：
Transfection efficiency of the immortalized human breast epithelial cell line MCF-10A remains an issue that needs to be resolved. In this study, it was aimed to deliver a recombinant DNA (pCMV-Azu-GFP) to the MCF-10A cells by the magnetofection method using magnetic nanoparticles (MNPs) and a simple magnet to accelerate the DNA delivery. Surface positively modified silica-coated iron oxide MNPs (MSNP-NH2) were produced and characterized via TEM, FTIR, and DLS analyses. The recombinant DNA (rDNA) was obtained by the integration of codon-optimized azurin to produce a fusion protein. Then, rDNA cloned in Escherichia coli cells was validated by sequence analysis. The electrostatically conjugated rDNA on MSNP-NH2 with an enhancer polyethyleneimine (PEI) was studied by agarose gel electrophoresis and the optimum conditions were determined to apply to the cell. A dose-dependent statistical difference was observed on treated cells based on the MTS test. The expression of the fusion protein after magnetofection was determined using laser scanning confocal microscope imaging and western blot analysis. It was observed that the azurin gene could be transferred to MCF-10A cells by magnetofection. Thus, when the azurin gene is used as a breast cancer treatment agent, it can be expressed in healthy cells without toxic effects.
摘要：
永生化人乳腺上皮细胞系MCF-10A的转染效率仍然是一个需要解决的问题。在这项研究中,其目的是使用磁性纳米颗粒(MNPs)和简单的磁体通过磁转染法将重组DNA(pCMV-Azu-GFP)递送至MCF-10A细胞以加速DNA递送。表面正改性二氧化硅包覆氧化铁MNPs（MSNP-NH2）的制备和表征通过TEM，FTIR,和DLS分析。通过整合密码子优化的天青蛋白以产生融合蛋白来获得重组DNA(rDNA)。然后，通过序列分析验证了在大肠杆菌细胞中克隆的rDNA。通过琼脂糖凝胶电泳研究了MSNP-NH2上与增强子聚乙烯亚胺（PEI）静电缀合的rDNA，并确定了适用于细胞的最佳条件。基于MTS测试,在处理的细胞上观察到剂量依赖性统计差异。使用激光扫描共聚焦显微镜成像和蛋白质印迹分析确定磁转染后融合蛋白的表达。观察到天青素基因可以通过磁转染转移到MCF-10A细胞中。因此，当天青蛋白基因被用作乳腺癌治疗剂时，它可以在健康细胞中表达而没有毒性作用。
