Losordo D W, Pickering J G, Takeshita S, Leclerc G, Gal D, Weir L, Kearney M, Jekanowski J, Isner J M
Department of Medicine (Cardiology), St Elizabeth's Hospital, Tufts University School of Medicine, Boston, Mass. 02135.
Circulation. 1994 Feb;89(2):785-92. doi: 10.1161/01.cir.89.2.785.
The development of molecular strategies for the treatment of restenosis has been hindered by low efficiencies of in vivo arterial transfection. Expression of intracellular marker proteins is generally evident in < 1% of vascular smooth muscle cells after in vivo arterial transfection. Efforts to improve the efficiency of in vivo gene transfer have been further impeded by the use of transgenes encoding for intracellular marker proteins, necessitating tissue removal and limiting survey for expression to one point in time.
To study gene expression on a serial basis in vivo and determine the relation between a secreted gene product and transfection efficiency after in vivo arterial gene transfer, a method for performing and serially monitoring gene expression in vivo was developed using the central artery of the rabbit ear. Liposome-mediated transfection of plasmid DNA containing the gene for human growth hormone (hGH) was successfully performed in 18 of 23 arteries. Serum hGH levels measured 5 days after transfection ranged from 0.1 to 3.8 ng/mL (mean, 0.97 ng/mL); in contrast, serum drawn from the control arteries demonstrated no evidence of hGH production. Serial measurement of hGH from transfected arteries demonstrated maximum hGH secretion 5 days after transfection and no detectable hormone after 20 days. Despite these levels of secreted gene product documented in vivo, immunohistochemical staining of sections taken from the rabbit ear artery at necropsy disclosed only rare cells in which there was evidence of successful transfection.
These experiments demonstrate a useful method of performing serial in vivo analyses of gene expression after vascular transfection and that anatomic analyses of transfection efficiency may underestimate the potential magnitude of expression in the case of a secreted gene product. These findings have implications for the clinical application of somatic gene therapy because low-efficiency transfection with a gene encoding for a secreted protein may achieve therapeutic effects not realized by transfection with genes encoding for proteins that remain intracellular.
体内动脉转染效率低下阻碍了用于治疗再狭窄的分子策略的发展。体内动脉转染后，细胞内标记蛋白的表达通常在不到1%的血管平滑肌细胞中明显可见。编码细胞内标记蛋白的转基因的使用进一步阻碍了提高体内基因转移效率的努力，这需要切除组织并将表达检测限制在一个时间点。
为了在体内连续研究基因表达，并确定体内动脉基因转移后分泌的基因产物与转染效率之间的关系，我们利用兔耳中央动脉开发了一种在体内进行并连续监测基因表达的方法。在23条动脉中的18条成功进行了脂质体介导的含人生长激素（hGH）基因的质粒DNA转染。转染后5天测得的血清hGH水平为0.1至3.8 ng/mL（平均0.97 ng/mL）；相比之下，从对照动脉抽取的血清未显示hGH产生的证据。对转染动脉的hGH进行连续测量显示，转染后5天hGH分泌量最大，20天后未检测到激素。尽管在体内记录到了这些分泌的基因产物水平，但尸检时从兔耳动脉获取的切片进行免疫组织化学染色显示，仅有罕见细胞有成功转染的证据。
这些实验证明了一种在血管转染后对基因表达进行连续体内分析的有用方法，并且在分泌基因产物的情况下，转染效率的解剖学分析可能低估了表达的潜在程度。这些发现对体细胞基因治疗的临床应用具有启示意义，因为用编码分泌蛋白的基因进行低效转染可能实现用编码保留在细胞内的蛋白的基因转染无法实现的治疗效果。
