中国组织工程研究

中国组织工程研究 ›› 2011, Vol. 15 ›› Issue (45): 8495-8498.doi: 10.3969/j.issn.1673-8225.2011.45.030

• 干细胞转基因表达 transgenic expression in stem cells • 上一篇    下一篇

骨髓间充质干细胞中突变型人低氧诱导因子1α真核表达载体的表达

张  男1，李  谌1，郭  韬1，刘丹平2   

  1. 辽宁医学院附属第一医院，1中心实验室，辽宁省医学组织工程重点实验室，辽宁省外科学重点实验室，2骨关节外科，辽宁省锦州市 121000
  • 收稿日期:2011-04-13 修回日期:2011-05-13 出版日期:2011-11-05 发布日期:2011-11-05
  • 通讯作者: 刘丹平，博士，教授，主任医师，硕士生导师，辽宁医学院附属第一医院骨关节外科，辽宁省锦州市 121000 liudanping2009@sohu.com
  • 作者简介:张男，男，1972年生，辽宁省锦州市人，汉族，1999年锦州医学院毕业，技师，主要从事骨组织工程实验技术研究。 youna1024@sina.com
  • 基金资助:

    辽宁省自然科学基金资助项目(20062199)。

Expression of mutant human hypoxia-inducible factor 1 alpha eukaryotic expression vector in bone marrow mesenchymal stem cells

Zhang Nan1, Li Chen1, Guo Tao1, Liu Dan-ping2   

  1. 1Department of Central Laboratory, First Affiliated Hospital of Liaoning Medical University, Jinzhou  121000, Liaoning Province, China; 2Department of Bone and Joint Surgery, First Affiliated Hospital of Liaoning Medical University, Jinzhou  121000, Liaoning Province, China
  • Received:2011-04-13 Revised:2011-05-13 Online:2011-11-05 Published:2011-11-05
  • Contact: Liu Dan-ping, Doctor, Professor, Chief Physician, Master’s supervisor, Department of Bone and Joint Surgery, First Affiliated Hospital of Liaoning Medical University, Jinzhou 121000, Liaoning Province, China liudanping2009@sohu.com
  • About author:Zhang Nan, Technician, Department of Central Laboratory, Liaoning Medical University, Jinzhou 121000, Liaoning Province, China youna1024@sina.com
  • Supported by:

    the Natural Science Foundation of Liaoning Province, No. 20062199*

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摘要：

背景：转基因小鼠体内实验证实，重组的低氧诱导因子1α可以促进皮肤形态功能正常的血管新生。
目的：构建能够在常氧条件下同时表达突变型低氧诱导因子1α目的蛋白和绿色荧光蛋白报告分子的新型腺病毒真核表达载体，并转染SD大鼠骨髓间充质干细胞，检测该基因在细胞中的表达情况。
方法：利用Lipofectamine 2000介导将构建成功的重组腺病毒真核表达载体pAd-HIF1αmu- IRES-hrGFP-1转染HEK293A细胞，包装病毒，以最佳感染指数=50将重组腺病毒转染大鼠骨髓间充质干细胞，并设3个对照组，即阳性对照组：转染Ad-CMV-HIF1α-IRES-hrGFP-1；阴性对照组：转染Ad-CMV-IRES-hrGFP-1组；空白组：未转染病毒。
结果与结论：①腺病毒载体成功转染HEK293A细胞，包装成功，细胞内有大量绿色荧光表达。②转染突变型低氧诱导因子1α腺病毒表达载体的细胞在常氧条件下蛋白表达量明显高于转其他3组，3个对照组间差异无显著性意义(P > 0.05)。提示突变型腺病毒真核表达载体Ad-HIF1α-IRES-hrGFP-1在HEK293A内成功包装；突变后低氧诱导因子1α基因能够在常氧条件下大量且高效表达。

关键词: 低氧诱导因子1&alpha, 基因突变, 重组腺病毒载体, 骨髓间充质干细胞, 转染

Abstract:

BACKGROUND: In vivo experiments of transgenic mice have shown that recombinant hypoxia-inducible factor 1 alpha (HIF-1α) can promote angiogenesis under the conditions of normal skin morphology and function.
OBJECTIVE: To construct a new adenovirus eukaryotic expression vector that can co-express HIF-1α mutant protein and human renilla reniformis green fluorescent protein (hrGFP) reporter molecule under normoxia conditions to transfect bone marrow mesenchymal stem cells (BMSCs) of the Sprague-Dawley rats. 
METHODS: A successful recombinant adenovirus eukaryotic expression vector pAd-HIF1αmu-IRES-hrGFP-1 was constructed and transfected into human embryonic kidney 293A cells (HEK293A) using Lipofectamine 2 000 for packaging the virus. The recombinant adenovirus eukaryotic expression vector was transfected into BMSCs of the rats at the best value multiplicity of infection 50. Three control groups were established: positive control group transfected with Ad-CMV-HIF1α-IRES-hrGFP-1, negative control group transfected with Ad-CMV-IRES-hrGFP-1 and blank control group without transfection.
RESULTS AND CONCLUSION: ①The adenovirus vectors were transfected into the HEK293A cells with packaging successfully and a large amount of green fluoresence expression was found in the cells. ②The protein expression in the cells transfected with mutant adenovirus HIF-1α expression vector under normoxia conditions was higher than that of the three control groups. The difference among the three control groups had no significance(P > 0.05). To transfect mutant adenovirus eukaryotic expression vector pAd-HIF1αmu-IRES-hrGFP-1 into the HEK293A cells with pakaging successfully can induce the great and efficient expression of HIF-1α gene under normoxia conditions.

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