中国组织工程研究 ›› 2015, Vol. 19 ›› Issue (32): 5140-5147.doi: 10.3969/j.issn.2095-4344.2015.32.011

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

LIM矿化蛋白1/低氧诱导因子1α慢病毒载体转染脂肪源性干细胞的成骨分化

潘玮敏1,刘 民2,杨建昌1,段春光2,黄 悦1   

  1. 1西安体育学院健康科学系,陕西省西安市  710068;
    2解放军第四军医大学全军骨科研究所,陕西省西安市  710032
  • 出版日期:2015-08-06 发布日期:2015-08-06
  • 作者简介:潘玮敏,女,1974年生,陕西省咸阳市人,汉族,2009年解放军第四军医大学毕业,博士,副教授,主要从事运动损伤治疗与康复的研究。
  • 基金资助:

    国家自然科学基金资助项目(81201409)

Osteogenic differentiation of adipose-derived stem cells transfected by lentivirus vector carrying LIM mineralization protein-1 and hypoxia-inducible factor-1alpha

Pan Wei-min1, Liu Min2, Yang Jian-chang1, Duan Chun-guang2, Huang Yue1   

  1. 1Department of Human Movement Studies, Xi’an Physical Education University, Xi’an 710068, Shaanxi Province, China; 
    2Institute of Orthopaedics and Traumatology, the Fourth Military Medical University, Xi’an 710032, Shaanxi Province, China
  • Online:2015-08-06 Published:2015-08-06
  • About author:Pan Wei-min, M.D., Associate professor, Department of Human Movement Studies, Xi’an Physical Education University, Xi’an 710068, Shaanxi Province, China
  • Supported by:

    the National Natural Science Foundation of China, No. 81201409

摘要:

背景:LIM矿化蛋白1及低氧诱导因子1α作为胞内蛋白,可分别从诱导成骨分化及促进血管再生两个方面促进成骨,联合二者高效诱导脂肪源性干细胞成骨分化具有重要的意义。
目的:探讨慢病毒载体介导的LIM矿化蛋白1及低氧诱导因子1α双基因转染的脂肪源性干细胞成骨分化的情况。
方法:应用反转录PCR技术克隆目的基因LIM矿化蛋白1及低氧诱导因子1α片段,并分别克隆于慢病毒骨架质粒pLVX-EF1α-DsRed-Hyg和pLVX-EF1α-IRES2-AcGFP1中,构建慢病毒载体主体质粒pLVX-EF1α-DsRed-Hyg-RLMP-1和pLVX-EF1α-AcGFP-RHIF-1α,随即与辅助质粒和包装质粒共转染Lenti-X 293T 细胞,包装慢病毒载体;采用组织块加酶消化法分离培养大鼠脂肪源性干细胞,使用流式细胞仪对其进行鉴定。分别在慢病毒转染脂肪源性干细胞 3,7,14 d后,应用反转录PCR 方法检测脂肪源性干细胞中成骨基因骨形态发生蛋白2、Runx-2、碱性磷酸酶、骨钙蛋白表达水平的同时检测血管内皮生长因子的表达水平。
结果与结论:pLVX-EF1α-DsRed-Hyg-RLMP-1和pLVX-EF1α-AcGFP-RHIF-1α可有效地转染入大鼠脂肪源性干细胞;反转录PCR结果显示成骨基因骨形态发生蛋白2、Runx-2、碱性磷酸酶、骨钙蛋白在LIM矿化蛋白1及低氧诱导因子1α双基因转染后第7天都开始明显过表达,并且可持续到14 d。说明LIM矿化蛋白1及低氧诱导因子1α双基因转染脂肪源性干细胞可提高其成骨活性。

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

关键词: 干细胞, 脂肪干细胞, 低氧诱导因子1α, 脂肪源性干细胞, 基因疗法, LIM矿化蛋白1, 慢病毒载体, 转染, 成骨分化, 国家自然科学基金

Abstract:

BACKGROUND: LIM mineralization protein-1 (LMP-1) and hypoxia-inducible factor-1α (HIF-1α) as intracellular proteins can induce osteogenic differentiation and promote angiogenesis, respectively. Therefore, their combination is of great significance for effectively inducing the osteogenic differentiation of adipose-derived stem cells.
OBJECTIVE: To study the osteogenic differentiation of adipose-derived stem cells transfected by lentivirus vector carrying LMP-1 and HIF-1α. 
METHODS: Reverse transcription-PCR technology was employed to clone LMP-1 and HIF-1α genes, and the genes were cloned to lentivirus vectors pLVX-EF1α-DsRed-Hyg and pLVX-EF1α-IRES2-AcGFP1 to construct main lentiviral vectors pLVX-EF1α-DsRed-Hyg-RLMP-1 and pLVX-EF1α-IRES2-AcGFP1-RHIF-1α. Then, Lenti-X 293T cells were transfected with main vectors pLVX-EF1α-DsRed-Hyg-RLMP-1 and pLVX-EF1α-IRES2-AcGFP1-RHIF-1α, packaging plasmid and coated plasmid. After that, lentiviral vectors were packaged to transfect adipose-derived stem cells from rats that were obtained by tissue explants culture and enzyme digestion methods. At 3, 7, 14 days after transfection, reverse transcription-PCR technology was adopted to detect the expression of osteogeic genes, such as bone morphogenetic protein 2, Runx-2, alkaline phosphatase, osteocalcin as well as to detect the expression of vascular endothelial growth factor.
RESULTS AND CONCLUSION: Lentiviral vectors pLVX-EF1α-DsRed-Hyg-RLMP-1 and pLVX-EF1α-IRES2-AcGFP1-RHIF-1α were effectively transfected into adipose-derived stem cells. Reverse transcription-PCR results showed that from the 7th day to the 14th day after lentivirus transfection, bone morphogenetic protein 2, Runx-2, alkaline phosphatase and osteocalcin all over-expressed. These findings indicate that the combination of LMP-1 and HIF-1α can enhance the osteogenic activity of adipose-derived 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

Key words: Transfection, Cell Differentiation, Viruses

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