中国组织工程研究

中国组织工程研究 ›› 2011, Vol. 15 ›› Issue (20): 3621-3624.doi: 10.3969/j.issn.1673-8225.2011.20.003

• 骨组织构建 bone tissue construction • 上一篇    下一篇

重组真核表达质粒pEGFP/hVEGF165转染兔成骨细胞条件的优化

陈国武，孟纯阳，蒋电明，胡侦明   

  1. 重庆医科大学附属第一院骨科，重庆市  400016
  • 收稿日期:2011-01-19 修回日期:2011-03-17 出版日期:2011-05-14 发布日期:2011-05-14
  • 通讯作者: 孟纯阳，博士，教授，重庆医科大学附属第一院骨科，重庆市 400016 Chunyangmeng 16@163.com
  • 作者简介:陈国武★，男，1984年生，四川省盐亭县人，汉族，重庆医科大学在读硕士，医师，主要从事脊柱外科方面的研究。 chenguowu5566@yahoo.com.cn
  • 基金资助:

    重庆市卫生局资助项目，编号：07-2-087。课题名称：新型纳米网孔n-HA/PA66/成骨细胞/VEGF组织工程人工骨修复骨缺损再血管化的实验研究。

Optimization of transfection conditions of recombinant plasmid pEGFP/hVEGF165 into rabbit osteoblasts in vitro

Chen Guo-wu, Meng Chun-yang, Jiang Dian-ming, Hu Zhen-ming   

  1. Department of Orthopedics, the First Affiliated Hospital of Chongqing Medical University,  Chongqing  400016, China
  • Received:2011-01-19 Revised:2011-03-17 Online:2011-05-14 Published:2011-05-14
  • Contact: Meng Chun-yang, Doctor, Professor, Department of Orthopedics, the First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China chunyangmeng16@ 163.com
  • About author:Chen Guo-wu★, Studying for master’s degree, Physician, Department of Orthopedics, the First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China chenguowu5566@ yahoo.com.cn
  • Supported by:

    the grant from Chongqing Health Bureau, No. 07-2-087*

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摘要：

背景：血管内皮生长因子能够提高骨细胞的增殖能力，促进骨折局部血管发生，在骨缺损修复过程中发挥着至关重要的作用。
目的：优化重组真核质粒pEGFP/hVEGF165转染兔成骨细胞的条件，为构建新型组织工程骨种子细胞提供实验基础。
方法：体外分离、培养并鉴定兔成骨细胞。分别应用0.8，1.0，1.2 μg的质粒和1.5，2.0，2.5，3.0 μL脂质体两两组合，通过脂质体法将重组真核表达质粒pEGFP/hVEGF165转染入兔成骨细胞，转染48 h后倒置荧光显微镜下观察并测定转染率。
结果与结论：随着脂质体剂量的增加，成骨细胞死亡增多。转染48 h后，倒置荧光显微镜下可见细胞质内发绿色荧光的小颗粒成弥散分布，或在核周浓集成块状。其中，脂质体2.5 μL和质粒1.2 μg条件下的转染率最高，可达56%。说明质粒与脂质体之间的交互作用能够显著影响转染率，脂质体2.5 μL和质粒1.2 μg是转染兔成骨细胞的最佳条件。

关键词: 血管内皮生长因子, 质粒, 脂质体, 成骨细胞, 转染, 组织工程骨

Abstract:

BACKGROUND: Vascular endothelial growth factor has an important role in bone repair by promoting angiogenesis, and by stimulating and affecting hemotaxis, proliferation, survival, and activity of major skeletal cells, including osteoblasts, osteoclasts, and chondrocytes.
OBJECTIVE: To optimize the conditions of transfection of recombinant pEGFP/hVEGF165 plasmid into rabbit osteoblasts by liposome and to provide experimental basis for constructing new seed cells for tissue engineered bone.
METHODS: Isolation, culture and identification of rabbit osteoblasts was conducted. Using different amounts and ratio of plasmid and liposome, in the 24-well plate, recombinant plasmid pEGFP/hVEGF165 was transfected into rabbit osteoblasts by liposome. Forty-eight hours after transfection, the transfention ratio was detected by inverted fluorescence microscope.
RESULTS AND CONCLUSION: The interaction between liposomes and plasmid could obviously affect transfention ratio. And under the conditions of plasmid liposome 2.5 μL and plasmid 1.2 μg in the 24-well culture plates, there was the highest transfection efficiency (56%). The optimal transfection conditions of recombinant plasmid pEGFP/hVEGF165 into rabbit osteoblasts in vitro were liposome 2.5 μL and plasmid 1.2 μg.

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