Rust E M, Westfall M V, Samuelson L C, Metzger J M
Department of Physiology, University of Michigan Medical School, Ann Arbor 48109-0622, USA.
In Vitro Cell Dev Biol Anim. 1997 Apr;33(4):270-6. doi: 10.1007/s11626-997-0046-x.
The main purpose of this study was to examine, for the first time, the ability of recombinant adenovirus to mediate gene transfer into cardiac myocytes derived from mouse embryonic stem (ES) cells differentiating in vitro. In addition, observations were made on the effect of adenovirus infection on cardiac myocyte differentiation and contractility in this in vitro system of cardiogenesis. ES cell cultures were infected at various times of differentiation with a recombinant adenovirus vector (AdCMVlacZ) containing the bacterial lacZ gene under the control of the cytomegalovirus (CMV) promoter. Expression of the lacZ reporter gene was determined by histochemical staining for beta-galactosidase activity. LacZ expression was not detected in undifferentiated ES cells infected with AdCMVlacZ. In contrast, infection of differentiating ES cell cultures showed increasing transgene expression with continued time in culture. Expression in ES-cell-derived cardiac myocytes was demonstrated by codetection of beta-galactosidase activity and troponin T with indirect immunofluorescence. At 24 h postinfection, approximately 27% of the cardiac myocytes were beta-galactosidase positive, and lacZ gene expression appeared to be stable for up to 21 d postinfection. Adenovirus infection had no apparent effect on the onset, extent, or duration of spontaneously contracting ES-cell-derived cardiomyocytes, indicating that cardiac differentiation and contractile function were not significantly altered in the infected cultures. The demonstration of adenovirus-mediated gene transfer into ES-cell-derived cardiac myocytes will aid studies of gene expression with this in vitro model of cardiogenesis and may facilitate future studies involving the use of these myocytes for grafting experiments in vivo.
本研究的主要目的是首次检测重组腺病毒介导基因转移至体外分化的小鼠胚胎干细胞（ES细胞）来源的心肌细胞的能力。此外，还观察了腺病毒感染对体外心脏发生系统中心肌细胞分化和收缩性的影响。在分化的不同时间，用含有在巨细胞病毒（CMV）启动子控制下的细菌lacZ基因的重组腺病毒载体（AdCMVlacZ）感染ES细胞培养物。通过对β-半乳糖苷酶活性进行组织化学染色来测定lacZ报告基因的表达。用AdCMVlacZ感染未分化的ES细胞未检测到lacZ表达。相反，感染分化的ES细胞培养物显示，随着培养时间的延长，转基因表达增加。通过间接免疫荧光共检测β-半乳糖苷酶活性和肌钙蛋白T，证实了ES细胞来源的心肌细胞中的表达。感染后24小时，约27%的心肌细胞β-半乳糖苷酶呈阳性，并且lacZ基因表达在感染后长达21天似乎是稳定的。腺病毒感染对自发收缩的ES细胞来源的心肌细胞的起始、程度或持续时间没有明显影响，表明在感染的培养物中心脏分化和收缩功能没有显著改变。腺病毒介导的基因转移至ES细胞来源的心肌细胞的证实，将有助于利用这种体外心脏发生模型进行基因表达研究，并可能促进未来涉及将这些心肌细胞用于体内移植实验的研究。
