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用于牙髓干细胞转染和分化的电穿孔法

Electroporation for transfection and differentiation of dental pulp stem cells.

作者信息

Rizk Ahmed, Rabie Bakr M

机构信息

Department of Orthodontics, Faculty of Dentistry, The University of Hong Kong , Hong Kong, China .

出版信息

Biores Open Access. 2013 Apr;2(2):155-62. doi: 10.1089/biores.2012.0273.

Abstract

Target gene delivery is needed to induce cellular differentiation or a specific therapeutic effect. Electroporation is a relatively safe and simple technique to deliver nucleic acids to the cell that acts by rendering cells transiently permeable using short periods of high voltage. In stem cell research, human dental pulp stem cells (hDPSCS) are highly accessible, and they exhibit broad differentiation potential. Until now, no studies have attempted to optimize electroporation parameters for DPSCs with respect to transfection efficiency and viability. In this study, we aimed to optimize transfection of DPSCs through varying different electroporation parameters, including voltage, mode of pulsation, and the number of pulses. As positive control, we used commonly utilized the chemical transfection reagents Lipofectamine 2000 and FuGene 6. In addition, we used our newly optimized transfection conditions to transfect hDPSCs with a functional chondrogenic transgene. We obtained higher transfection efficiency and cell viability with these electroporation conditions compared to controls. The highest transfection efficiency (63.81±4.72%) was achieved with 100 V, 20 msec, one-pulse square-wave condition. Among chemical transfection groups, FuGene 6 showed the highest cell viability at all tested transfection ratios, while Lipofectamine 2000 showed the highest transfection efficiency (19.23±3.19%) using 1:1 DNA (μg):Lipofectamine (μL). Transfected DPSCs functionally expressed the transforming growth factor β-3 chondrogenic transgene on the mRNA level as detected by real-time polymerase chain reaction and on the protein level as detected by Western blot analysis. An increase in various chondrogenic markers was also found when studying mRNA expression in transfected cells. In conclusion, the results of our study demonstrate optimal electroporation and chemical transfection reagent conditions for hDPSCs, and, subsequently, we provide proof of concept for expression of a functional gene using those conditions. These results demonstrate a widened scope for use of DPSCs in various tissue engineering applications.

摘要

需要进行靶基因递送以诱导细胞分化或产生特定的治疗效果。电穿孔是一种相对安全且简单的将核酸递送至细胞的技术,其作用原理是利用短时间的高电压使细胞瞬时通透。在干细胞研究中,人牙髓干细胞(hDPSCs)易于获取,并且具有广泛的分化潜能。到目前为止,尚无研究针对转染效率和活力对牙髓干细胞的电穿孔参数进行优化。在本研究中,我们旨在通过改变不同的电穿孔参数,包括电压、脉冲模式和脉冲数,来优化牙髓干细胞的转染。作为阳性对照,我们使用了常用的化学转染试剂Lipofectamine 2000和FuGene 6。此外,我们使用新优化的转染条件,用功能性软骨生成转基因转染人牙髓干细胞。与对照组相比,在这些电穿孔条件下我们获得了更高的转染效率和细胞活力。在100 V、20毫秒、单脉冲方波条件下实现了最高的转染效率(63.81±4.72%)。在化学转染组中,FuGene 6在所有测试的转染比例下均显示出最高的细胞活力,而Lipofectamine 2000在使用1:1 DNA(μg):Lipofectamine(μL)时显示出最高的转染效率(19.23±3.19%)。通过实时聚合酶链反应检测,转染的牙髓干细胞在mRNA水平上功能性表达转化生长因子β-3软骨生成转基因,通过蛋白质印迹分析检测在蛋白质水平上也有表达。在研究转染细胞中的mRNA表达时,还发现各种软骨生成标志物有所增加。总之,我们的研究结果证明了人牙髓干细胞的最佳电穿孔和化学转染试剂条件,随后,我们提供了使用这些条件表达功能性基因的概念验证。这些结果表明牙髓干细胞在各种组织工程应用中的使用范围得到了拓宽。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/9e21/3620497/f73714c401b4/fig-1.jpg

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