上海交通大学学报（医学版）

›› 2011, Vol. 31 ›› Issue (8): 1078-.doi: 10.3969/j.issn.1674-8115.2011.08.006

• 专题报道（眼科视觉科学） • 上一篇    下一篇

腺相关病毒介导色素上皮衍生因子转染人虹膜色素上皮细胞的体外研究

赵 莼1,2, 王 方2, 计 菁1, 施沃栋1, 范先群1, 罗 敏1   

  1. 1.上海交通大学 医学院附属第九人民医院眼科, 上海 200011； 2.同济大学 医学院附属第十人民医院眼科, 上海 200072
  • 出版日期:2011-08-28 发布日期:2011-08-29
  • 通讯作者: 罗 敏, 电子信箱: qiangson@sh163.net。
  • 作者简介:赵 莼（1979—）, 女, 主治医师, 硕士；电子信箱: chunour@163.com。
  • 基金资助:

    上海市卫生局基金（2006028）

Adeno-associated virus mediated transduction of pigment epithelium-derived factor to human iris pigment epithelial cells in vitro

ZHAO Chun1,2, WANG Fang2, JI Jing1, SHI Wo-dong1, FAN Xian-qun1, LUO Min1   

  1. 1.Department of Ophthalmology, the Ninth People's Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200011, China；2.Department of Ophthalmology, the Tenth People's Hospital, Tongji University, Shanghai 200072, China
  • Online:2011-08-28 Published:2011-08-29
  • Supported by:

    Shanghai Municipal Health Bureau Foundation, 2006028

PDF (PC)

1575

摘要：

目的 探讨以腺相关病毒（AAV）为载体，对体外培养的人虹膜色素上皮（IPE）细胞进行色素上皮衍生因子（PEDF）转染，获得高效表达PEDF转基因细胞的可行性。方法 构建携带PEDF基因的重组AAV载体AAV-PEDF，体外培养人IPE，用1×106 pfu的AAV-PEDF按感染复数(MOI)为0、2、10和20分别感染体外培养的IPE细胞；荧光显微镜下观察细胞绿色荧光蛋白表达情况；流式细胞术检测细胞转染效率；RT-PCR和Western blotting法鉴定PEDF在IPE细胞中的表达。结果 AAV-PEDF转染后，IPE细胞生长正常，用荧光显微镜、RT-PCR和Western blotting均检测到PEDF表达。在MOI为20时，细胞感染阳性率达64.22%。结论 AAV-PEDF能有效地转染体外培养的IPE细胞并有效表达PEDF。

关键词: 腺相关病毒, 色素上皮衍生因子, 虹膜色素上皮细胞, 转染

Abstract:

Objective To investigate the feasibility of recombinant adeno-associated virus (AAV) mediated transduction of pigment epithelium-derived factor (PEDF) to cultured human iris pigment epithelial (IPE) cells in vitro to obtain the transgenic cells with high expression of PEDF. Methods Recombinant AAV vector carrying PEDF gene (AAV-PEDF) was constructed, and human IPE cells were cultured in vitro and infected with condensed virus. The expression of green fluorescent protein in cells was observed under fluorescence microscopy, the transfection efficiency was detected by flow cytometry, and the expression of PEDF in IPE cells was detected by RT-PCR and Western blotting. Results The proliferation of IPE cells was normal after infection with AAV-PEDF. The expression of PEDF was observed under fluorescence microscopy and detected by RT-PCR and Western blotting. When the multiplicity of infection (MOI) was 20, the transduction efficiency was 64.22%. Conclusion AAV-PEDF can effectively transfect IPE cells cultured in vitro, and the transgenic cells have a high expression of PEDF.

Key words: adeno-associated virus, pigment epithelium-derived factor, iris pigment epithelial cell, transduction