Enhancement of adenoviral gene transfer to adult rat cardiomyocytes in vivo by immobilization and ultrasound treatment of the heart
Enhancement of adenoviral gene transfer to adult rat cardiomyocytes in vivo by immobilization and ultrasound treatment of the heart
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DOI：
10.1038/sj.gt.3302476
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发表时间：
2005-06-01
期刊：
影响因子：
5.1
通讯作者：
Fuller, SJ
中科院分区：
文献类型：
--
作者：
Sato, M;O'Gara, P;Fuller, SJ
将腺病毒载体直接注射到体内心室心肌中产生包括心肌细胞在内的细胞的局部转染。使用与目的基因共表达GFP的载体允许随后鉴定几天后从心脏分离的转染的肌细胞，并在细胞浴实验中检查它们的功能。我们已经将受磷蛋白的反义载体或仅表达GFP的对照病毒注射到体内成年大鼠心脏中，然后在7天后取出心脏并分离心室肌细胞。在注射期间和注射后使用止血夹短暂固定心室，使转染的杆状活肌细胞的数量从1.7 +/- 0.8%（n = 8）增加到5.6 +/- 0.8%（n = 9）。通过在注射前后向该部位施加超声脉冲，这进一步增加至13.2 +/- 1.1%（n = 8）。Phospholamban反义增加了转染心肌细胞的收缩幅度，加速了心肌细胞的再充盈或Ca 2+瞬变的下降，而GFP对照则没有。受磷蛋白下调的定性和定量作用在体内和体外转染之间是相当的。这种技术将有许多用途，包括生产转染的心肌细胞，而没有培养诱导的收缩性变化的问题。
Direct injection of adenoviral vectors into ventricular myocardium in vivo produces local transfection of cells including cardiomyocytes. The use of vectors coexpressing GFP with the gene of interest allows subsequent identification of transfected myocytes isolated from the heart some days later, and examination of their function in cell bath experiments. We have injected vectors for antisense to phospholamban, or a control virus for expression of GFP only, into adult rat heart in vivo and then removed the heart and isolated ventricular myocytes 7 days later. Brief immobilization of the ventricle during and after injection using a haemoclip increased the number of transfected rod-shaped, viable myocytes from 1.7 +/- 0.8% ( n = 8) to 5.6 +/- 0.8% ( n = 9). This was further increased to 13.2 +/- 1.1% ( n = 8) by the application of ultrasound pulses to the site before and after injection. Phospholamban antisense increased contraction amplitude and accelerated myocyte relengthening or decline of the Ca2+ transient in transfected myocytes, while GFP control did not. Qualitative and quantitative effects of phospholamban down-regulation were comparable between in vivo and in vitro transfections. This technique will have a number of uses, including production of transfected myocytes without the problem of culture-induced changes in contractility.