中国组织工程研究

中国组织工程研究 ›› 2010, Vol. 14 ›› Issue (7): 1239-1243.doi: 10.3969/j.issn.1673-8225.2010.07.023

• 组织构建基础实验 basic experiments in tissue construction • 上一篇    下一篇

脂质体介导化学合成siRNA转染原代心肌细胞：筛选理想浓度

李  杰，贾钰华，杨  萍，周凤华，李丽君   

  1. 南方医科大学中医药学院，广东省广州市  510515
  • 出版日期:2010-02-12 发布日期:2010-02-12
  • 通讯作者: 贾钰华，硕士，教授，博士生导师，主任医师，南方医科大学中医药学院， 广东省广州市 510515 jyh@fimmu.com
  • 作者简介:李 杰☆，男，1982年生，山西省晋中市人，汉族，南方医科大学在读博士，主要从事血管疾病的中医药防治研究。 ztxlj@qq.com
  • 基金资助:

    课题受国家自然基金项目（30572435）“心肌缺血及再灌注心律失常的蛋白质组基础与定心方作用机制”资助。

Liposomes-mediated chemosynthesis siRNA transfection to primary cardiomyocytes: Selection of an ideal concentration

Li Jie, Jia Yu-hua, Yang Ping, Zhou Feng-hua, Li Li-jun   

  1. School of Traditional Chinese Medicine, Southern Medical University, Guangzhou  510515, Guangdong Province, China
  • Online:2010-02-12 Published:2010-02-12
  • Contact: Jia Yu-hua, Master, Professor, Doctoral supervisor, Chief physician, School of Traditional Chinese Medicine, Southern Medical University, Guangzhou 510515, Guangdong Province, China jyh@fimmu.com
  • About author:Li Jie☆, Studying for doctorate, School of Traditional Chinese Medicine, Southern Medical University, Guangzhou 510515, Guangdong Province, China ztxlj@qq.com
  • Supported by:

     the National Natural Science Foundation of China, No.30572435*

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摘要：

背景：siRNA转染是完成RNA干扰的关键一步。目前用于心肌细胞的RNA干扰技术多以质粒为载体转染长链shRNA，其步骤复杂。脂质体转染化学合成短链siRNA是一种操作简便，低毒高效的转染方法，将其应用于心肌细胞转染，有利于RNA干扰技术的推广应用。
目的：筛选脂质体介导化学合成siRNA转染原代心肌细胞的最佳浓度，以及简单高效的RNA干扰操作方法。
方法：应用脂质体siPORTTM NeoFXTM转染试剂介导5，10，20，30 nmol/L的CY3-Negative siRNA转染心肌细胞，同时设立空白对照组，转染24 h后分别应用荧光显微镜，流式细胞仪检测转染效率和细胞凋亡率，筛选最优浓度。根据优化浓度转染PHB siRNA，48 h后检测蛋白表达验证转染效果。
结果与结论：随CY3-Negative siRNA浓度的增加，在荧光显微镜下观察到激发出红色荧光的细胞比例随之增加，流式细胞仪检测出各组平均转染效率也依次增高(P < 0.05)，其中30 nmol/L组转染效率最高(P < 0.05)，而各实验组与空白对照组之间的细胞凋亡率差异无显著性意义(P > 0.05)。将心肌细胞转染30 nmol/L 的PHB siRNA，48 h后PHB蛋白表达平均下降74.11%(P < 0.05)。实验结果表明，脂质体转染试剂siPORTTM NeoFXTM介导化学合成siRNA转染原代心肌细胞的理想浓度是30 nmol/L。

关键词: 脂质体, siRNA, 转染, 心肌细胞, 心脏组织工程

Abstract:

BACKGROUND: siRNA transfection is a key step in RNA interference. The methods of cardiomyocytes transfection were most use of plasmids as vector to transfect long-chain shRNA. However, the processes were complex. It was a simple efficient and low-toxic method that liposomes-mediated chemosynthesis siRNA transfection. It was useful for expanding RNA interference application.
OBJECTIVE: To choice the optimal concentration of liposomes-mediated chemosynthesis siRNA transfection, and to discover a simple efficient RNA interference application.
METHODS: CY3-Negative siRNA was mediated by lipid-based agent siPORTTM NeoFXTM to transfect cardiomyocytes. A blank control group was set. After 24 hours, the transfection efficiency and apoptotic rate were evaluated by fluorescent microscope and flow cytometer to select an optimal concentration. Based the best concentration, siRNA PHB was transfected to cardiomyocytes. 48 hours later, the expression of PHB was tested.
RESULTS AND CONCLUSION: With the increased concentration of CY3-Negative siRNA, the number of cells emitted red fluorescence grew under fluorescence microscope, and the transfection efficiency was also increased (P < 0.05). The best concentration was 30 nmol/L (P < 0.05). There was no significant difference in apoptotic rate between transfected groups and the control group (P > 0.05). The PHB expression of cardiomyocytes transfected siRNA PHB was dropped by 74.11% on average (P < 0.05). The results indicated that lipid based agent siPORTTM NeoFXTM was suitable to transfect chemosynthesis siRNA to cardiomyocytes，and the best transfection concentration of siRNA was 30 nmol/L.

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