| 周 明,李 浩,王子建,李登科,全正扬,孙震晓.常规脂质体介导shRNA转染建立CYP2E1表达沉默的人肝实质细胞模型[J].,2018,(1):18-22 |
| 常规脂质体介导shRNA转染建立CYP2E1表达沉默的人肝实质细胞模型 |
| Construction of Conventional Cationic Liposome Interfering Cytochrome P450 2E1-silence Human Hepatic Parenchymal Cell Line |
| 投稿时间：2017-03-16 修订日期：2017-04-12 |
| DOI：10.13241/j.cnki.pmb.2018.01.004 |
| 中文关键词: 脂质体转染 CYP2E1 shRNA 人肝实质细胞 G418 qRT - PCR |
| 英文关键词: Cationic liposome transfection Cytochrome P450 2E1 Short hairpin RNA Human hepatic parenchymal cells G418 qRT-PCR |
| 基金项目:国家自然科学基金项目(81473418)；国家林业局野生动植物保护项目(2012-2016)；北京中医药大学东直门医院"111"协同创新院际合作项目（No.2016-DZM111-ZY008） |
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| 中文摘要: |
| 摘要 目的：利用常规脂质体介导转染shRNA（short hairpin RNA）建立CYP2E1（cytochrome P450 2E1）表达沉默的人肝实质细胞模型。方法：采用文献报道的CYP2E1的高效干扰位点，构建CYP2E1的shRNA干扰载体（shCYP2E1）以及同源无干扰作用的对照载体（shNC，non-specific control），并通过阳离子脂质体LipoFiterTM介导转染人肝实质L02 细胞，通过绿色荧光蛋白报告基因的表达指示及G418筛选阳性克隆，qRT-PCR (quantitative reverse transcription polymerase chain reaction) 测定稳定转染细胞系中CYP2E1的表达情况。结果：确定转染后G418最佳筛选浓度为400 μg/mL，维持浓度为200 μg/mL；获得了荧光表达率较高的L02-CYP2E1细胞（CYP2E1沉默组）和L02-NC细胞（转染对照组）；qRT-PCR结果显示，所建细胞系L02-CYP2E1相对于L02-NC，其CYP2E1表达下调了近70 % (P<0.05)，表明CYP2E1干扰表达细胞模型构建成功。结论：利用常规脂质体转染法成功转染人肝实质L02细胞CYP2E1shRNA，建立了CYP2E1表达沉默的细胞系。 |
| 英文摘要: |
| ABSTRACT Objective: To build a CYP2E1-silence human hepatic parenchymal cell line by conventional liposome-mediated RNA interference technology. Methods: Fluorescence labeling interference vector of CYP2E1 (shCYP2E1) and nonsense vector (shNC, non-specific control) were constructed on the reported efficient interfering sequence. These vectors were transfected into human hepatic parenchymal L02 cells by the cationic liposome LipoFiterTM. Positive cell clones were screened by G418 and expression of green fluo- rescent protein reporting gene. Expression of CYP2E1 of these screened cells were determined by qRT-PCR. Results: The optimum se- lection concentration of G418 was 400 μg/mL and the maintenance concentration was 200 μg/mL. Stable transfected L02-CYP2E1 cells(transfected with vector of CYP2E1) and L02-NC cells(transfected with nonsense vector) were obtained. The qRT-PCR data showed that the CYP2E1 expression in L02-CYP2E1 cells reduced approximately 70 % to L02-NC cells (P<0.05), which suggested that L02 cell model with CYP2E1-silence was constructed successfully. Conclusion: CYP2E1-silence human hepatic parenchymal cell line was suc- cessfully constructed by conventional liposome-mediated RNA interference technology. |
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