中国组织工程研究

中国组织工程研究 ›› 2010, Vol. 14 ›› Issue (36): 6695-6698.doi: 10.3969/j.issn.1673-8225.2010.36.010

• 脂肪干细胞 adipose-derived stem cells • 上一篇    下一篇

携带增强绿色荧光蛋白的慢病毒载体感染人脂肪源性干细胞的有效性

王巍巍，张金元   

  1. 解放军第四五五医院肾脏科，南京军区肾脏专科中心，上海市 200052
  • 出版日期:2010-09-03 发布日期:2010-09-03
  • 通讯作者: 张金元，主任医师，教授，博士后导师，解放军第四五五医院肾脏科，上海市 200052 jinyuan_zhang@163.com
  • 作者简介:王巍巍☆，女，1975年生，黑龙江省哈尔滨市人，汉族，2007年解放军南京军区南京总医院博士后出站，博士，副主任医师，硕士生导师，主要从事干细胞在肾损伤中的应用研究。 w.vwei@163.com
  • 基金资助:

    上海市青年科技启明星计划基金(09QA1407500)项目，上海市自然科学基金(08ZR1423000)项目。

Transfection of human adipose-derived stem cells with lentiviral vector containing enhanced-green fluorescent protein: A validity study

Wang Wei-wei, Zhang Jin-yuan   

  1. Department of Nephrology, the 455 Hospital of Chinese PLA, Nephrology Center of Nanjing Military Area Command of Chinese PLA, Shanghai  200052, China
  • Online:2010-09-03 Published:2010-09-03
  • Contact: Zhang Jin-yuan, Chief physician, Professor, Doctoral supervisor, Department of Nephrology, the 455 Hospital of Chinese PLA, Nephrology Center of Nanjing Military Area Command of Chinese PLA, Shanghai 200052, China jinyuan_zhang@163.com
  • About author:Wang Wei-wei☆, Doctor, Associate chief physician, Master’s supervisor, Department of Nephrology, the 455 Hospital of Chinese PLA, Nephrology Center of Nanjing Military Area Command of Chinese PLA, Shanghai 200052, China w.vwei@163.com
  • Supported by:

    the Youth Technology Venus Project of Shanghai City, No. 09QA1407500*; the Natural Science Foundation of Shanghai City, No. 08ZR1423000*

PDF

403

被引次数

5

摘要：

背景：细胞移植前，将目的基因转染干细胞可增强细胞治疗效果。但要实现目的基因的高效转移并适度表达，载体系统的选择是关键。
目的：将携带绿色荧光蛋白的慢病毒载体感染人脂肪源性干细胞，检测病毒感染效率及感染后的细胞活力。
方法：利用脂质体转染法获取慢病毒原液。设定不同感染复数(1，5，10，15，20)，将带有增强绿色荧光蛋白的慢病毒载体感染人脂肪源性干细胞。荧光显微镜下观察病毒转染后人脂肪源性干细胞内增强绿色荧光蛋白的表达，流式细胞仪测定病毒感染效率。茜素红染色及油红O染色鉴定病毒转染后人脂肪源性干细胞的多向分化潜能。MTT法检测转染后人脂肪源性干细胞的增殖情况。
结果与结论：随病毒感染时间及感染复数值的增加，人脂肪源性干细胞内绿色荧光表达明显增强，感染后第3天，感染复数为20时人脂肪源性干细胞内可见明显绿色荧光表达，病毒感染效率为60%；第5~7天，病毒感染效率可达90%~95%。转染后人脂肪源性干细胞经成骨及成脂诱导液培养后，茜素红染色及油红O染色阳性，表明病毒感染未影响人脂肪源性干细胞的多向分化能力。病毒感染后，人脂肪源性干细胞的增殖能力未受明显影响。说明携带增强绿色荧光蛋白的慢病毒载体可以有效感染人脂肪源性干细胞。

关键词: 慢病毒载体, 人脂肪源性干细胞, 转染, 增强绿色荧光蛋白, 细胞活力

Abstract:

BACKGROUND: Before transplantation, the ability of stem cells was obviously increased by the transfection of target gene. The key of gene therapy is that target gene was transferred effectively and was expressed appropriately in stem cells. The choice of carrier system will affect the treatment effectiveness. OBJECTIVE: To observe the transfection efficiency of lentiviral vector transfecting the human adipose-derived stem cells (hASCs) and to detect the vitality of hASCs transfected.

METHODS: The recombinant lentiviral supernatant was obtained by standard lipofectamine reagent. The hASCs were transfected by lentiviral vector containing enhanced-green fluorescent protein (EGFP) with different multiplicity of infection (MOI=1, 5, 10, 15, 20). Transfection efficiency was assessed by flow cytometer for EGFP expression under fluorescence microscope. The multi-directional differentiation of hASCs after transfection was evaluated by alizarin red staining and oil red O staining. The proliferation of hASCs after transfection was detected by MTT assay. RESULTS AND CONCLUSION: Transfection efficiency was 60% at 3 day and 90%-95% at 5-7 day after infection under MOI=20. The alizarin red staining and oil red O staining showed hASCs after transfected hASCs were induced to osteogenic and adipogenic lineages, which suggested that viral infection could not affect multi-directional differentiation of hASCs. The proliferation of transfected hASCs had no marked change. This showed that hASCs transfected by lentiviral vectors carrying EGFP can effectively infect hASCs.

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