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

在这项研究中，我们描述了一种优化的方案，电穿孔为基础的转染支持细胞，并比较其效率与传统的脂质体转染。采用方波电穿孔法在不同条件下将pCMV-GFP质粒转染Sertoli细胞。根据细胞存活率和表达EGFP的百分比，确定250 V产生最大数量的瞬时转染细胞。在电压保持不变（250 V）的情况下，脉冲宽度为20μm时细胞存活率（76.5 ± 3.4%）和转染效率（30.6 ± 5.6%）相对较高。随着质粒DNA浓度的增加（10-50μg/ml），阳性细胞的产量从14.0 ± 2.8%增加到35.0 ± 6.3%;而质粒DNA浓度为20μg/ml时，细胞存活率从73.1 ± 4.9%下降到57.0 ± 6.6%。本文介绍了电穿孔条件的优化过程，并成功地将质粒DNA电穿孔到原代培养的睾丸支持细胞中。结果表明，电穿孔法更适合于睾丸支持细胞的转染。

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.