《口腔颌面外科杂志》 ›› 2012, Vol. 22 ›› Issue (5): 323-327. doi: 10.3969/j.issn.1005-4979.2012.05.004

• 基础研究 • 上一篇    下一篇

低氧诱导因子-1α慢病毒载体的构建及其在骨髓基质干细胞内表达的检测

王 瀛1,张凯2,邹多宏1,何家才1,王元银1,周健1   

  1. 1. 安徽医科大学口腔医学院口腔颌面外科,安徽 合肥230032;2.蚌埠医学院附属第一人民医院口腔科,安徽 蚌埠233000
  • 出版日期:2012-10-28 发布日期:2013-01-02
  • 通讯作者: 周健,教授. E-mail: zj@ahmu. edu. cn
  • 作者简介:王瀛(1986—),女,贵州大方人,硕士研究生. E-mail:229379088@163.com
  • 基金资助:

     国家自然科学基金资助项目(81100788,81070864);安徽省自然科学基金资助项目 (11040606M173, 11040606M204);安徽医科大学博士科研启动基金资助项目 (XJ201109, XJ201034)

Construction of Lentiviral Vector Carrying HIF-1α  and Detection of  the Target Gene Expression in BMSCs

WANG Ying1, ZHANG Kai2, ZOU Duo-hong1, HE Jia-cai1, WANG Yuan-yin1, ZHOU Jian1   

  1. 1.Department of Oral and Maxillofacial Surgery, College of Stomatology, Anhui Medical University, Hefei, 230032; 2.Department of Stomatology, The First People’s Hospital, Bengbu Medical College, Bengbu 233000, Anhui Province, China
  • Online:2012-10-28 Published:2013-01-02

摘要: 目的:构建低氧诱导因子-1α(HIF-1α)的慢病毒载体(Lenti-HIF-1α),转染骨髓基质干细胞 (bone marrow stromal cells, BMSCs) 后,检测HIF-1α在BMSCs 内的表达,并鉴定其位置。方法:根据人的HIF-1α基因(NM_001530)序列行引物设计及序列片段的PCR扩增,将目的基因 PCR 产物连接到载体 pEGFP-N1 上,构建真核表达载体 pEGFP-N1-HIF-1α。将目的基因PCR产物和目的载体用NheI和BamHI分别进行酶切,对质粒进行鉴定。采用 LR 重组系统将目的序列重组到慢病毒载体 plenti6.3V5-DEST上,构建慢病毒载体Lenti-HIF-1α (Lenti-LacZ为对照组)。检测慢病毒滴度后,转染 BMSCs,检测目的基因的表达。通过细胞免疫组织化学法观察 HIF-1α 在 BMSCs 内的位置。结果:通过对构建质粒克隆进行测序及酶切,证实真核表达载体pEGFP-N1-HIF-1α构建成功。用Lenti-HIF-1α转染BMSCs细胞,0、1、4、7、14和21 d后qPCR检测。结果表明 HIF-1α 在转染后的第4天开始有明显的过表达,且持续到第21天。细胞免疫组织化学结果显示目的基因位于BMSCs的核内。结论:成功构建了慢病毒载体Lenti-HIF-1α,且转染的目的基因位于BMSCs细胞核内,为HIF-1α介导的 BMSCs进行体内实验研究奠定了基础。

关键词: 低氧诱导因子1&alpha, ;慢病毒载体;骨髓间充质干细胞

Abstract: Objective: The study was designed to construct Lenti-HIF-1α, and detect HIF-1α expression and location in bone marrow mesenchymal stem cells (BMSCs) transduced by Lenti-HIF-1α. Methods: According to human HIF-1α gene sequence (NM_001530), its primer was designed and was amplified through PCR. The eukaryotic expression vector (pEGFP-N1-HIF-1α) was constructed by connecting the PCR products of the target gene to the vector pEGFP-N1. To identify the plasmid, target gene PCR product and the purpose vector were digested by NheI and BamHI. Lenti-HIF-1α (control group, Lenti-LacZ) was constructed using the LR recombination system (the lentiviral vector plenti6.3V5-DEST). After lentiviral titer was detected, BMSCs was transduced by Lenti-HIF-1α. The analysis of target gene expression was done with qPCR. Besides, the immunohistochemistry examination was also completed to observe the location of HIF-1α in BMSCs. Results: The results of plasmid sequencing and digestion confirmed that the eukaryotic expression vector pEGFP-N1-HIF-1α was successfully constructed. After Lenti-HIF-1α was transduced to BMSCs at 0 d, 1 d, 4 d, 7 d, 14 d and 21 d, the results of qPCR showed that the over-expression of HIF-1α was detected on 4 d, and continued until the 21 d. Immunohistochemical results showed that the target gene was located in the nucleus of BMSCs. Conclusion: We successfully constructed the Lenti-HIF-1α, and target gene located in the nucleus of BMSCs. This study will lay the foundation for experimental studies of bone defect repair using HIF-1α-mediated BMSCs in the future.

Key words:  hypoxia inducible factor 1α, lentiviral vector, bone marrow mesenchymal stem cells

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