中国组织工程研究

中国组织工程研究 ›› 2014, Vol. 18 ›› Issue (1): 57-62.doi: 10.3969/j.issn.2095-4344.2014.01.010

• 骨髓干细胞 bone marrow stem cells • 上一篇    下一篇

低氧诱导因子1α慢病毒载体转染骨髓间充质干细胞的成骨基因表达

张劲娥1，2，王淑红1，3，张  忻1，郭华艳2，郭  娜1，黄远亮2   

  1. 1同济大学口腔医学院，上海市  200072
    2同济大学附属东方医院口腔科，上海市  200120
    3杭州市第一人民医院口腔科，浙江省杭州市  310006
  • 修回日期:2013-10-17 出版日期:2014-01-01 发布日期:2014-01-01
  • 通讯作者: 黄远亮，男，教授，博士生导师，同济大学附属东方医院口腔科，上海市 200120
  • 作者简介:张劲娥，女，1985年生，同济大学口腔医学院在读硕士，主要从事口腔医学方面的研究。 并列第一作者：王淑红，女，1976年生，同济大学口腔医学院在读博士，主治医师，主要从事口腔临床医学方面的研究。
  • 基金资助:

    国家自然科学基金重点项目( 81070806)；浙江省自然科学基金(LQ12H14002)；上海市科委医学重点课题(10JC1413300)；杭州市医药卫生科技计划项目(2012A015)

Influence of hypoxia-inducible factor-1 alpha lentiviral vector on osteogenic gene expression of rabbit bone marrow mesenchymal stem cells

Zhang Jin-e1, 2, Wang Shu-hong1, 3, Zhang Xin1, Guo Hua-yan2, Guo Na1, Huang Yuan-liang2   

  1. 1School of Stomatology, Tongji University, Shanghai 200072, China
    2Department of Stomatology, Shanghai East Hospital Affiliated to Tongji University, Shanghai 200011, China
    3Department of Stomatology, Hangzhou First People’s Hospital, Hangzhou 310006, Zhejiang Province, China
  • Revised:2013-10-17 Online:2014-01-01 Published:2014-01-01
  • Contact: Huang Yuan-liang, Professor, Doctoral supervisor, Department of Stomatology, Shanghai East Hospital Affiliated to Tongji University, Shanghai 200011, China
  • About author:Zhang Jin-e, Studying for master’s degree, School of Stomatology, Tongji University, Shanghai 200072, China; Department of Stomatology, Shanghai East Hospital Affiliated to Tongji University, Shanghai 200011, China Wang Shu-hong, Studying for doctorate, Attending physician, School of Stomatology, Tongji University, Shanghai 200072, China; Department of Stomatology, Hangzhou First People’s Hospital, Hangzhou 310006, Zhejiang Province, China Zhang Jin-e and Wang Shu-hong contributed equally to this work.
  • Supported by:

    the National Natural Science Foundation of China, No. 81070806; the Natural Science Foundation of Zhejiang Province, No. LQ12H14002; Medical Key Topics of Shanghai Science and Technology Commission, No. 10JC1413300; the Medicine and Health Science and Technology Projects in Hangzhou, No. 2012A015

PDF

601

摘要：

背景：成骨作用和血管发生在骨的形成和修复过程中紧密结合，而低氧诱导因子1α被认为是促血管新生相关基因调控中最重要的核心转录因子，其可促进缺氧部位血管的形成，进而促进骨的形成。
目的：构建野生型和点突变型两个低氧诱导因子1α慢病毒真核表达载体Lenti-HIF1α-IRES-EGFP，并比较其对兔骨髓间充质干细胞成骨基因表达的影响。
方法：首先根据野生型人源低氧诱导因子1α基因序列信息和确定酶切位点的点突变型序列信息构建低氧诱导因子1α基因野生型和点突变型两个慢病毒真核表达载体；然后采用制备的病毒液转染兔骨髓间充质干细胞。
结果与结论：免疫荧光显微镜观察显示，转染7 d后野生型组和点突变型组细胞均未见明显荧光，转染14 d后两组细胞均呈现明显的绿色荧光；qPCR定量分析结果表明，转染7 d后即有低氧诱导因子1α和骨形态发生蛋白2基因的显著表达，转染14 d后，两基因仍然显示较高的表达水平。说明实验成功构建野生型和点突变型低氧诱导因子1α基因慢病毒真核表达载体，转染后可以促进兔骨髓间充质干细胞成骨基因的表达。


中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程


全文链接：

关键词: 干细胞, 骨髓干细胞, 低氧诱导因子1α, 点突变, 慢病毒, 转染, 骨髓间充质干细胞, 基因治疗, 聚合酶链反应, 细胞学实验, 骨形态发生蛋白2, 国家自然科学基金

Abstract:

BACKGROUND: Osteogenesis is closely integrated with angiogenesis in bone formation and repair process, and hypoxia-inducible factor 1 alpha (HIF-1α) is considered to be the most important core transcription factor promoting angiogenesis gene regulation, which may promote the formation of blood vessels at hypoxia portion, and thus contribute to bone formation.
OBJECTIVE: To construct the Lenti-HIF-1α-IRES-EGFP (wild type) and Lenti-HIF-1α-IRES-EGFP (point mutant type) lentiviral eukaryotic expression vectors and to detect their impact on the osteogenic gene expression of rabbit bone marrow mesenchymal stem cells.
METHODS: The Lenti-HIF-1α-IRES-EGFP (wild type) and Lenti-HIF-1α-IRES-EGFP (point mutant type) lentiviral eukaryotic expression vectors were constructed according to the wild type human HIF-1α gene sequence and the determined restriction sites of human HIF-1α point mutant sequence. Rabbit bone marrow mesenchymal stem cells were transfected with the prepared Lenti-HIF-1α-IRES-EGFP (wild type) and Lenti-HIF-1α-IRES-EGFP (point mutant type) virus solution.
RESULTS AND CONCLUSION: Immunofluorescence microscopy observations indicated that the cells of Lenti-HIF-1α-IRES-EGFP (wild type) group and Lenti-HIF-1α-IRES-EGFP (point mutant type) group showed no obvious fluorescence on transfected 7 days, and two groups of cells showed a more obvious green fluorescent after transfection 14 days. Quantitative PCR analysis results showed that there were obvious HIF-1α and bonemorphogenetic protein 2 gene expressions on days 7 after transfection and the two genes still showed highly expression levels after transfection 14 days. The two lentiviral eukaryotic expression vectors of Lenti-HIF-1α-IRES-EGFP (wild type) and Lenti-HIF-1α-IRES-EGFP (point mutant type) could be constructed according to the wild type human HIF-1α gene sequence and the determined restriction sites of human HIF-1α point mutant sequence; HIF-1α gene can promote the osteogenic gene expression of lentivirus-transfected rabbit bone marrow mesenchymal stem cells.


中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程


全文链接：

Key words: lentivirus, mesenchymal stem cells, bone marrow, bone morphogenetic proteins, viruses, transfection

中图分类号: