马粉娜, 林国生, 曾彬. 腺病毒介导hHO-1基因体外转染骨髓间充质干细胞[J]. 心脏杂志, 2009, 21(2): 174-178.
    引用本文: 马粉娜, 林国生, 曾彬. 腺病毒介导hHO-1基因体外转染骨髓间充质干细胞[J]. 心脏杂志, 2009, 21(2): 174-178.
    Experimental study on bone marrow mesenchymal stem cell transfected by Adv-HO-1[J]. Chinese Heart Journal, 2009, 21(2): 174-178.
    Citation: Experimental study on bone marrow mesenchymal stem cell transfected by Adv-HO-1[J]. Chinese Heart Journal, 2009, 21(2): 174-178.

    腺病毒介导hHO-1基因体外转染骨髓间充质干细胞

    Experimental study on bone marrow mesenchymal stem cell transfected by Adv-HO-1

    • 摘要: 目的 以腺病毒(adenovirus,Adv)为载体将人血红素加氧酶(human hemo-oxygenase,hHO)-1基因转染至骨髓间充质干细胞(MSC)中,为干细胞移植联合hHO-1基因治疗心血管疾病奠定实验基础。方法 以密度梯度离心结合贴壁筛选法分离培养骨髓MSC。将携带有hHO-1基因与绿色荧光蛋白(GFP)报告基因的腺病毒体外扩增与鉴定后,以不同感染复数(multiplicity of infection,MOI)转染MSC。在荧光显微镜下观察转染后GFP的表达,以反转录(RT)-聚合酶链式反应(PCR)法检测目的基因hHO-1 mRNA在MSC中的表达,4’6’-二乙酰基-2-苯基吲哚(DAPI)染色观察hHO-1基因转染对MSC的影响。结果 将Adv-hHO-1体外扩增、纯化后,经PCR鉴定可得到555 bp的目的条带。转染后24 h,MSC中即可见GFP的表达,荧光强度随MOI的增加而增强。RT-PCR法检测显示,不同MOI的转染组均有hHO-1 mRNA表达,随MOI的增加其表达强度也逐渐增强(P<0.05)。DAPI染色显示,hHO-1基因转染后,细胞的生长及形态无明显变化。结论 以Adv为载体可成功地将hHO-1基因转染至骨髓MSC中,转染效率随转染滴度的增加而增加,转染后对细胞的生物学活性无明显影响。

       

      Abstract: AIM To transfect human hemo-oxygenase-1(hHO-1) gene into bone marrow Mesenchymal stem cell(MSC) via adenovirus (Adv) vector, so as to lay the experimental foundation for the therapy of cardiovascular disease. METHODS Bone marrow-derived MSC were separated and purified in vitro by Percoll density gradient centrifugation and screened by cell adhered to the bottom. MSC were transfected with different multiplicity of infection (MOI) of Adv-hHO-1-green fluorescent protein(GFP) after amplification, purification and identification. GFP expression was examined with fluorescence microscope and hHO-1 mRNA expression was observed with reverse transcription(RT)-polymerase chain reaction(PCR). 4’,6’-amidino-2’-phenylindol(DAPI) staining was used to observe the morphology of MSC after transfection. RESULTS PCR result showed a band of 555 bp DNA after Adv-GFP-hHO-1 amplification and purification. GFP was observed after 24 h transfection and the intensity of GFP expression enhanced in parallel with MOI. RT-PCR showed that the HO-1 mRNA expressed in different transfection groups and the expression also elevated in parallel with MOI. DAPI staining showed no obvious changes in the morphology of MSC. CONCLUSION hHO-1 gene is successfully transfected into MSC via Adv vector, which exerts no obvious adverse influence on the growth and proliferation of MSC. The transfection efficiency increases along with the increase of MOI.

       

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