Smith Jeffrey S, Xu Zhili, Byrnes Andrew P
Division of Cellular and Gene Therapies, Food and Drug Administration, Center for Biologics Evaluation and Research, Bethesda, MD 20892, USA.
J Virol Methods. 2008 Jan;147(1):54-60. doi: 10.1016/j.jviromet.2007.08.009. Epub 2007 Sep 11.
Kupffer cells are a major barrier to systemic adenovirus (Ad) gene therapy because they rapidly and efficiently clear virions from the circulation. The lack of a straightforward quantitative technique for selectively measuring uptake of Ad by Kupffer cells has made it difficult to study the mechanisms by which they recognize Ad. A new method was developed that relies on immunofluorescent detection of Ad within Kupffer cells in mouse liver sections, followed by confocal microscopy and computerized image analysis. The method is sensitive, quantitative and reproducible, with a linear range spanning two orders of magnitude. As an example of the utility of this method, it was found that pre-injecting mice with polyinosinic acid reduces accumulation of Ad in Kupffer cells by approximately 90%.
库普弗细胞是全身腺病毒（Ad）基因治疗的主要障碍，因为它们能迅速且高效地从循环中清除病毒颗粒。缺乏一种直接的定量技术来选择性测量库普弗细胞对Ad的摄取，使得研究它们识别Ad的机制变得困难。开发了一种新方法，该方法依赖于对小鼠肝脏切片中库普弗细胞内Ad的免疫荧光检测，随后进行共聚焦显微镜检查和计算机图像分析。该方法灵敏、定量且可重复，线性范围跨越两个数量级。作为该方法实用性的一个例子，发现预先给小鼠注射聚肌苷酸可使库普弗细胞中Ad的积累减少约90%。
