重组腺病毒缺氧诱导因子-1α体外靶向性转染大鼠缺氧脑微血管内皮细胞的研究

In vitro targeted transfection of recombinant adenovirus vectors containing the hypoxia inducible factor-1αgene in rat hypoxic brain microvascular endothelial cells

目的探讨重组腺病毒缺氧诱导因子-1α(AdHIF-α1)在大鼠缺氧脑微血管内皮细胞(BMEC)中的转染情况,为AdHIF-1α治疗缺氧的BMEC提供理论基础。方法建立大鼠BMEC的体外分离培养及鉴定,并用含100μmol/L二氯化钴(CoCl2)的维持培养液培养,制成BMEC缺氧模型;然后将AdHIF-1α转染入缺氧的BMEC中,在荧光显微镜下观察荧光蛋白的转染情况。结果AdHIF-lα转染入缺氧的BMEC后,分别在12 h、24 h、48 h、72 h于荧光显微镜下观察AdHIF-lα的转染情况,结果显示,12 h开始出现少许荧光,光密度值(A值)0.13±0.01;24 h荧光表达有所增强,A值0.46±0.03;48 h荧光表达最明显,A值0.97±0.05;72 h可见荧光减弱,A值0.38±0.02;不同时间点比较差异有统计学意义,两两比较结果显示,相邻时间点A值比较差异均有统计学意义(q=25.88、40.00、46.28,均P<0.01)。结论重组腺病毒缺氧诱导因子-1α能成功转染至缺氧的BMEC。

Objective To investigate the transfection of recombinant adenovirus vectors containing the hypoxia inducible factor-1αgene(AdHIF-1α)in rat brain microvascular endothelial cells(BMECs)and to provide a theoretical basis for the treatment of hypoxic BMECs by AdHIF-1α.Methods Rat BMECs were isolated,identified,and cultured in a maintenance medium containing 100μmol/L cobalt dichloride(CoCl2),establishing a hypoxia model of BMECs;then AdHIF-1αwas transfected into hypoxic BMECs.The transfection of fluorescent protein was observed under a fluorescence microscope.Results Transfection of AdHIF-1αinto BMECs was monitored under a fluorescence microscope at 12 h,24 h,48 h and 72 h,respectively.Minor fluorescence began to appear at 12 h(0.13±0.01),and the fluorescence expression increased at 24 h(0.46±0.03,q=25.88,P<0.01),was most obvious at 48 h(0.97±0.05,q=40.00,P<0.01),and decreased at 72 h(0.38±0.02,q=46.28,P<0.01).Conclusions Recombinant adenovirus vectors containing AdHIF-1αcan be transfected into hypoxic BMECs in vitro.