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| 电穿孔法转染人原代成纤维细胞条件的优化 |
| 张放1,2,周载鑫1,张丹丹3,房贺4,刘善荣1* |
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(1. 第二军医大学长海医院实验诊断科, 上海 200433;
2. 解放军东部战区73901部队, 上海 200439;
3. 第二军医大学长海医院临床实验中心, 上海 200433;
4. 第二军医大学长海医院烧伤科, 上海 200433
*通信作者) |
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| 摘要: |
| 目的 通过优化电穿孔转染条件，探讨影响人原代成纤维细胞电转染效率和转染后细胞存活率的因素，筛选出人原代成纤维细胞的最佳电转染条件。方法 通过改变脉冲电压、脉冲次数和质粒浓度等条件，将含有增强型绿色荧光蛋白（EGFP）基因的质粒pEGFP-N1电穿孔转染入人原代成纤维细胞。利用锥虫蓝染色检测细胞存活情况，采用流式细胞术检测转染24 h后的转染效率。结果 在低电压模式下，脉冲电压320 V、质粒浓度为20 μg/mL、脉冲1次时，人原代成纤维细胞的电转染效果最理想，转染效率达到（42.53±0.63）%，细胞存活率为（74.30±3.01）%。结论 优化了人原代成纤维细胞的电转染条件，在保证细胞较高存活率的前提下提高了人原代成纤维细胞的电转染效率，为后续基因功能的研究打下基础。 |
| 关键词: 电穿孔 成纤维细胞 细胞存活 转染效率 脉冲 |
| DOI：10.16781/j.0258-879x.2017.10.1311 |
| 投稿时间：2017-03-25修订日期：2017-06-29 |
| 基金项目:国家自然科学基金（81172472），中国博士后科学基金（2016M593004），东方学者跟踪支持计划（GZ2015009）. |
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| Optimization of transfection conditions of human primary fibroblasts by electroporation |
| ZHANG Fang1,2,ZHOU Zai-xin1,ZHANG Dan-dan3,FANG He4,LIU Shan-rong1* |
(1. Department of Laboratory Medicine, Changhai Hospital, Second Military Medical University, Shanghai 200433, China;
2. No. 73901 Troop of PLA Eastern Theater Command, Shanghai 200439, China;
3. Clinical Laboratory Center, Changhai Hospital, Second Military Medical University, Shanghai 200433, China;
4. Department of Burn Surgery, Changhai Hospital, Second Military Medical University, Shanghai 200433, China
*Corresponding author) |
| Abstract: |
| Objective To investigate the factors affecting the success rate and cell survival rate of electroporation-mediated transfection of human primary fibroblasts by optimizing the electroporation parameters, so as to screen the optimal conditions for electroporation-mediated transfection of human primary fibroblasts. Methods Plasmid pEGFP-N1 containing enhanced green fluorescent protein (EGFP) gene was transfected into human primary fibroblasts with electroporation by changing the conditions such as pulse voltage, pulse number and plasmid concentration. Cell mortality rate was detected by trypan blue staining. Transfection efficiency was measured by flow cytometry at 24 h after transfection. Results The electroporation-mediated transfection effect of human primary fibroblasts was optimal under the following condition:low voltage mode, pulse voltage of 320 V, plasmid concentration of 20 μg/mL and single pulse, with the cell transfection efficiency being (42.53±0.63)% and the cell survival rate being (74.30±3.01)%. Conclusion The electroporation-mediated transfection efficiency of primary human fibroblasts can be improved by optimizing the transfection conditions, which might lay a foundation for subsequent function research of gene. |
| Key words: electroporation fibroblasts cell survival transfection efficiency pulse |
