Establishment of human Sertoli cell line and analysisof molecular mechanism of testicular dysfunction induced by anticancer drug

在这项研究中，我们描述了一种基于电穿孔的Sertoli细胞转染的优化方案，并将其效率与传统的脂质体转染法进行了比较。用方波电穿孔法在不同条件下将pCMV-GFP载体导入Sertoli细胞。根据细胞存活率和表达EGFP的百分率，确定250V可产生最大数量的瞬时转染细胞。当电压恒定(250V)时，20μm的脉冲具有较高的细胞存活率(76.5±3.4%)和转染率(30.6±5.6%)。在10~50μg/ml浓度范围内，阳性细胞产率随浓度的增加而增加，从14.0±2.8%增加到35.0±6.3%，而细胞存活率在2 0μg/ml时从73.1±4.9%下降到5 7.0±6.6%。我们描述了优化电穿孔条件的过程，并成功地将质粒DNA电穿孔到原代培养的Sertoli细胞中。结果表明，电穿孔法更适合于Sertoli细胞的转染法。

In this study, we described an optimized protocol for electroporation-based transfection of Sertoli cells and compared its efficiency with conventional lipofection. Sertoli cells were transfected with pCMV-GFP plasmid by square-wave electroporation in different conditions. According for both cell survival and the percentage expressing EGFP, 250 V was determined to produce the greatest number of transiently transfected cells. Keeping voltage consistent (250 V), the pulse length of 20μm was observed relatively higher cell survival (76.5 ± 3.4%) and transfection efficiency (30.6 ± 5.6%). The yield of positive cells increased with increasing concentrations of plasmid DNA (range 10-50μg/ml) from 14.0 ± 2.8% to 35.0 ± 6.3% respectively, but cells viability steadily decreased following 20μg/ml plasmid DNA from 73.1 ± 4.9% to 57.0 ± 6.6%. We described the process of optimizing electroporation conditions, and the successful electroporation of plasmid DNA into primarily cultured Sertoli cells. Our results indicated that the method of electroporation is more suitable for transfection of Sertoli cells.