Zhou Y Y, Wang S Q, Zhu W Z, Chruscinski A, Kobilka B K, Ziman B, Wang S, Lakatta E G, Cheng H, Xiao R P
Laboratory of Cardiovascular Science, Gerontology Research Center, National Institute on Aging, National Institutes of Health, Baltimore, Maryland 21224, USA.
Am J Physiol Heart Circ Physiol. 2000 Jul;279(1):H429-36. doi: 10.1152/ajpheart.2000.279.1.H429.
Rapid development of transgenic and gene-targeted mice and acute genetic manipulation via gene transfer vector systems have provided powerful tools for cardiovascular research. To facilitate the phenotyping of genetically engineered murine models at the cellular and subcellular levels and to implement acute gene transfer techniques in single mouse cardiomyocytes, we have modified and improved current enzymatic methods to isolate a high yield of high-quality adult mouse myocytes (5.3 +/- 0.5 x 10(5) cells/left ventricle, 83.8 +/- 2.5% rod shaped). We have also developed a technique to culture these isolated myocytes while maintaining their morphological integrity for 2-3 days. The high percentage of viable myocytes after 1 day in culture (72.5 +/- 2.3%) permitted both physiological and biochemical characterization. The major functional aspects of these cells, including excitation-contraction coupling and receptor-mediated signaling, remained intact, but the contraction kinetics were significantly slowed. Furthermore, gene delivery via recombinant adenoviral infection was highly efficient and reproducible. In adult beta(1)/beta(2)-adrenergic receptor (AR) double-knockout mouse myocytes, adenovirus-directed expression of either beta(1)- or beta(2)-AR, which occurred in 100% of cells, rescued the functional response to beta-AR agonist stimulation. These techniques will permit novel experimental settings for cellular genetic physiology.
转基因小鼠和基因靶向小鼠的快速发展，以及通过基因转移载体系统进行的急性基因操作，为心血管研究提供了强大的工具。为了便于在细胞和亚细胞水平对基因工程小鼠模型进行表型分析，并在单个小鼠心肌细胞中应用急性基因转移技术，我们改进并完善了当前的酶法，以分离出高产率的高质量成年小鼠心肌细胞（5.3±0.5×10⁵个细胞/左心室，83.8±2.5%为杆状）。我们还开发了一种技术，用于培养这些分离的心肌细胞，同时在2至3天内保持其形态完整性。培养1天后存活心肌细胞的高百分比（72.5±2.3%）使得对其进行生理和生化特性分析成为可能。这些细胞的主要功能方面，包括兴奋-收缩偶联和受体介导的信号传导，仍然完好无损，但收缩动力学明显减慢。此外，通过重组腺病毒感染进行基因递送效率高且可重复。在成年β₁/β₂-肾上腺素能受体（AR）双敲除小鼠心肌细胞中，腺病毒介导的β₁-或β₂-AR的表达（在100%的细胞中发生）挽救了对β-AR激动剂刺激的功能反应。这些技术将为细胞遗传生理学提供新的实验环境。
