QUANTITATIVE-DETERMINATION OF ADENOVIRUS-MEDIATED GENE DELIVERY TO RAT CARDIAC MYOCYTES IN-VITRO AND IN-VIVO
QUANTITATIVE-DETERMINATION OF ADENOVIRUS-MEDIATED GENE DELIVERY TO RAT CARDIAC MYOCYTES IN-VITRO AND IN-VIVO
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DOI：
10.1073/pnas.90.24.11498
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发表时间：
1993-12-15
影响因子：
11.1
通讯作者：
LEINWAND, LA
中科院分区：
文献类型：
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作者：
KASSEISLER, A;FALCKPEDERSEN, E;LEINWAND, LA
为了优化使用修饰的腺病毒作为载体的基因传递到心肌，我们已经表征了感染培养的胎儿和成年大鼠心肌细胞在体外和成年心肌细胞在体内使用的复制缺陷型腺病毒携带氯霉素乙酰转移酶（CAT）报告基因驱动的巨细胞病毒启动子（AdCMVCATgD）。在体外，当每个细胞感染1个噬斑形成单位的病毒时，几乎所有的胎儿或成人心肌细胞都表达CAT基因。CAT酶活性可以在这些细胞中检测到早在感染后4小时，在48小时达到接近最大水平。在胎儿细胞中，CAT表达保持至少1周的活动没有损失。以体外研究为指导，我们将AdCMVCATgD病毒直接导入成年大鼠心肌，并将病毒注射获得的表达结果与直接注射pAdCMVCATgD质粒DNA获得的表达结果进行比较。由腺病毒感染心肌引起的CAT活性的量比从DNA注射中看到的高几个数量级，并且与输入病毒的量成比例。在腺病毒注射后的心脏组织切片中CAT蛋白的免疫染色显示大量阳性细胞，在心脏的许多区域中几乎达到100%的肌细胞。由腺病毒引入的基因表达在感染后5天达到峰值，但在感染后55天仍可检测到。因此，腺病毒是一个非常有用的工具，高效率的基因转移到心血管系统。
To optimize the use of modified adenoviruses as vectors for gene delivery to the myocardium, we have characterized infection of cultured fetal and adult rat cardiac myocytes in vitro and of adult cardiac myocytes in vivo by using a replication-defective adenovirus carrying the chloramphenicol acetyltransferase (CAT) reporter gene driven by the cytomegalovirus promoter (AdCMVCATgD). In vitro, virtually all fetal or adult cardiocytes express the CAT gene when infected with 1 plaque-forming unit of virus per cell. CAT enzymatic activity can be detected in these cells as early as 4 hr after infection, reaching near-maximal levels at 48 hr. In fetal cells, CAT expression was maintained without a loss in activity for at least 1 week. Using in vitro studies as a guide, we introduced the AdCMVCATgD virus directly into adult rat myocardium and compared the expression results obtained from virus injection with those obtained by direct injection of pAdCMVCATgD plasmid DNA. The amount of CAT activity resulting from adenovirus infection of the myocardium was orders of magnitude higher than that seen from DNA injection and was proportional to the amount of input virus. Immunostaining for CAT protein in cardiac tissue sections following adenovirus injection demonstrated large numbers of positive cells, reaching nearly 100% of the myocytes in many regions of the heart. Expression of genes introduced by adenovirus peaked at 5 days but was still detectable 55 days following infection. Adenoviruses are therefore a very useful tool for high-efficiency gene transfer into the cardiovascular system.