基础医学与临床 ›› 2014, Vol. 34 ›› Issue (3): 365-371.

• 研究论文 • 上一篇    下一篇

HIF-1α转染方法的优化及其对间充质干细胞存活及分化功能的影响

张扬,辛畅泰   

  1. 沈阳医学院附属奉天医院
  • 收稿日期:2013-01-24 修回日期:2013-10-07 出版日期:2014-03-05 发布日期:2014-02-27
  • 通讯作者: 张扬 E-mail:paulzhang_1979@hotmail.com

Method optimization of HIF-1αtransfection and its effects on survival rate and differentiation of mesenchymal stem cells

  • Received:2013-01-24 Revised:2013-10-07 Online:2014-03-05 Published:2014-02-27

摘要: 目的 探索应用脂质体作为载体将低氧诱导因子-1α(hif-1α)转染至间充质干细胞(MSCs)的转染方法。方法 应用脂质体作为载体将HIF-1α转染至P3代MSCs,荧光观察绿色荧光蛋白(GFP)表达,评估转染效率。比较在不同情况下细胞的转染效率。将转染的MSCs、空质粒转染的MSCs和未转染的MSCs置于缺氧环境下(CoCl2模拟缺氧),比较3种细胞HIF-1αmRNA、HIF-1α蛋白的表达。鉴定转染的MSCs向成骨细胞和脂肪细胞的分化能力,及在缺氧环境中的存活率。结果 应用脂质体法可以成功将pcDNA3.0-HIF-1α-eGFP转染至MSCs。当细胞汇合度在80%以上,DNA与质粒的比为(μg/nl)1:1.25,且转染时间为4h时转染效率最高,为21%。转染HIF-1α的MSCs在缺氧环境中可被成功向脂肪细胞和成骨细胞诱导分化,HIF-1α转染可以提高缺氧时MSCs存活率(P <0.05)。在缺氧条件下pcDNA3.0-HIF-1α-eGFP瞬时转染的MSCs较空质粒(pcDNA3.0-eGFP)转染的MSCs和未转染的MSCs能高表达HIF-1αmRNA及HIF-1α蛋白(P <0.05)。结论 应用脂质体作为载体可以将HIF-1α转染至MSCs,HIF-1α转染的MSCs在缺氧时高表达HIF-1αmRNA及蛋白。

关键词: 骨髓间充质干细胞, di氧诱导因子-1, 脂质体, 转染

Abstract: Object To investigate a method of MSCs transfection with HIF-1αgene-recombined liposome. Method HIF-1αwas transferred into the third generation MSCs by liposome. The expression of green flouresence protein (GFP) was observed and the efficiency of transfection was assessed. The three groups of cells, which are MSCs transfected with HIF-1αgene-recombined liposome, with control vector or with no transfection, were cultured in the hypoxia environment simulated through CoCl2. The expressions of HIF-1αmRNA, HIF-1αprotein of the different groups of MSCs were compared. Result pcDNA3.0-HIF-1α-eGFP could be transfected into MSCs by the liposome method successfully and the efficiency of the transfection is 21%. MSCs transfected with HIF-1α can be induced to differentiate to osteoblast and lipoblast successfully and HIF-1αtransfection may increase MSCs survival rate under hypoxia condition(P <0.05).The group of HIF-1α-transfected cells express much more HIF-1αmRNA and HIF-1α protein than the other two(P <0.05). Conclusion The study shows that the liposome can be used to transfect HIF-1α into MSCs successfully and MSCs transfected by HIF-1α gene-recombined liposome can strongly express HIF-1α mRNA and HIF-1α protein.

Key words: mesenchymal stem cell(MSC), hypoxia inducible factor-1(hif-1), liposo-me, transfection

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