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

人支持细胞系的建立及抗癌药物引起睾丸功能障碍的分子机制分析

• 批准号: 23791757
• 负责人: YAMAGUCHI Kohei
• 金额: $ 1.66万
• 依托单位: Kobe University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Young Scientists (B)
• 财政年份: 2011
• 资助国家: 日本
• 起止时间: 2011 至 2012
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-23791757/
• 关键词: セルトリ細胞; エレクトロポレーション; トランスフェクション; EGFP; hTERT

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.

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20 μ m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（10-50 μ g/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20 μ g/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。

项目成果

精子形成：基礎と臨床の接点 精子形成機構の理解に基づいた男性不妊診療

精子发生：基础知识与临床实践之间的接口基于对精子发生机制的理解的男性不育症治疗

• 发表时间: 2011
• 作者: Li Fuping;Yamaguchi Kohei;Okada Keisuke;Matsushita Kei;Enatsu Noritoshi;Chiba Koji;Yue Huanxun;Fujisawa Masato.;Li Fuping;Li Fuping;Fuping Li;山口 耕平
• 通讯作者: 山口 耕平

icient transfection of DNAinto primarily cultured rat sertoli cellsby electroporation

电穿孔法将DNA快速转染至原代培养的大鼠支持细胞中

• 发表时间: 2013
• 作者: Li Fuping;Yamaguchi Kohei;Okada Keisuke;Matsushita Kei;Enatsu Noritoshi;Chiba Koji;Yue Huanxun;Fujisawa Masato.;Li Fuping
• 通讯作者: Li Fuping

Efficient Transfection of DNA into Primarily Cultured Rat Sertoli Cells by Electroporation

• DOI: 10.1095/biolreprod.112.106260
• 发表时间: 2013-03-01
• 期刊: BIOLOGY OF REPRODUCTION
• 影响因子: 3.6
• 作者: Li, Fuping;Yamaguchi, Kohei;Fujisawa, Masato
• 通讯作者: Fujisawa, Masato