中国组织工程研究

中国组织工程研究 ›› 2010, Vol. 14 ›› Issue (14): 2588-2590.doi: 10.3969/j.issn.1673-8225.2010.14.024

• 干细胞基础实验 basic experiments of stem cells • 上一篇    下一篇

Ad5-增强型绿色荧光蛋白和rAAV2-增强型绿色荧光蛋白转染脂肪间充质干细胞的对比

袁小洪1，安荣泽1，王兆杰1，贾婀娜2，齐新文1，陈金平1，杨  晋1，刘凡凡1   

  1. 1遵义医学院第五附属(珠海)医院，广东省珠海市  519100；2遵义医学院珠海校区，广东省珠海市  519000
  • 出版日期:2010-04-02 发布日期:2010-04-02
  • 作者简介:袁小洪★，男，1980年生，重庆市人，汉族，遵义医学院珠海校区在读硕士，医师，主要从事组织工程软骨的研究。 dahai809@sina.com
  • 基金资助:

    贵州省省长基金资助课题(黔科通NY字[2006]62-2)。

Ad5-enhanced green fluorescent protein versus rAAV2-enhanced green fluorescent protein transfecting adipose-derived mesenchymal stem cells  

Yuan Xiao-hong1, An Rong-ze1, Wang Zhao-jie1, Jia E-nuo2, Qi Xin-wen1, Chen Jin-ping1, Yang Jin1, Liu Fan-fan1   

  1. 1 Fifth Affiliated (Zhuhai) Hospital, Zunyi Medical College, (Zhuhai)   519100, Guangdong Province, China; 2 Zhuhai Campus, Zunyi Medical College, Zhuhai   519000, Guangdong Province, China
  • Online:2010-04-02 Published:2010-04-02
  • About author:Yuan Xiao-hong★, Studying for master’s degree, Physician, Fifth Affiliated (Zhuhai) Hospital, Zunyi Medical College, Zhuhai 519100, Guangdong Province, China dahai809@sina.com
  • Supported by:

    the Nomarch Foundation of Guizhou Province, No. Qianketong NY zi [2006]62-2*

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摘要：

背景：利用基因转染技术诱导脂肪间充质干细胞成软骨细胞已有报道，但腺病毒和腺相关病毒转染脂肪间充质干细胞各有不同，且腺相关病毒载体能否转染脂肪间充质干细胞众说不一。
目的：观察Ad5-增强型绿色荧光蛋白(enhanced green fluorescent protein，EGFP)和rAAV2-EGFP转染脂肪间充质干细胞后增强型绿色荧光蛋白的表达及转染后细胞增殖能力的变化。
方法：脂肪组织来源于6月龄新西兰大白兔颈背部。机械消化和酶消化法分离提取脂肪间充质干细胞，体外培养扩增。分别采用腺病毒载体(Ad5-EGFP)和腺相关病毒载体(rAAV2-EGFP)转染脂肪间充质干细胞，并观察EGFP的表达。rAAV2-EGFP转染后，需加入2 μL丁酸钠(1 mol/L)。采用MTT法检测基因转染对脂肪间充质干细胞增殖能力的影响。
结果与结论：体外培养的脂肪间充质干细胞呈扁平状和长梭形，细胞形态均一，传代稳定。Ad5-EGFP组和rAAV2-EGFP组均能观察到较多荧光细胞，转染效率分别达到88%和83%。腺病毒和腺相关病毒载体均能转染脂肪间充质干细胞，且转染效率很高。腺相关病毒需要借助丁酸钠来提高其基因表达水平。

关键词: 腺病毒, 腺相关病毒, 脂肪间充质干细胞, 增强型绿色荧光蛋白, 转染

Abstract:

BACKGROUND: There are reports concerning differentiation of adipose-derived mesenchymal stem cells (ADSCs) into chondrocytes using gene transfection technique. However, the transfection of adenovirus and adeno-associated virus into ADSCs is various. It is controversial whether adeno-associated virus (AAV) can transfect ADSCs.
OBJECTIVE: To observe the enhanced green fluorescent protein (EGFP) expression following Ad5-EGFP and rAAV2-EGFP transfection into ADSCs, and investigate the cell proliferation ability following transfection.
METHODS: ADSCs were isolated from the adipose tissue, which was from 6-month-old New Zealand albino rabbit back and neck by mechanical digestion and enzyme digestion, then ADSCs were cultured and amplified in vitro. ADSCs were infected with Ad5-EGFP and rAAV2-EGFP, and the EGFP expression was observed. A total of 2 μL sodium butyrate (1 mol/L) was added into the medium after rAAV2-EGFP transfection. MTT assay was used to detect the gene transfection effects on reproductive activity of ADSCs.
RESULTS AND CONCLUSION: ADSCs isolated and cultured in vitro were flat, long-spindle and amplified stably. The cell morphology was uniform. Many green fluorescent cells were observed in Ad5-EGFP and rAAV2-EGFP groups. Transfection efficiencies were about 88% and 83%. Adenovirus and adeno-associated virus vector can be transfected with ADSCs, and transfection efficiency is high. Adeno-associated virus needs sodium butyrate to increase its level of gene expression.

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