Division of Cardiovascular Diseases, Department of Internal Medicine, Mayo Clinic, Rochester, MN 55905, USA.
JACC Cardiovasc Imaging. 2013 Jul;6(7):795-802. doi: 10.1016/j.jcmg.2012.11.018. Epub 2013 May 1.
The goal of this study was to validate a pathway-specific reporter gene that could be used to noninvasively image the oxidative status of progenitor cells.
In cell therapy studies, reporter gene imaging plays a valuable role in the assessment of cell fate in living subjects. After myocardial injury, noxious stimuli in the host tissue confer oxidative stress to transplanted cells that may influence their survival and reparative function.
Rat mesenchymal stromal cells (MSCs) were studied for phenotypic evidence of increased oxidative stress under in vitro stress. On the basis of their up-regulation of the pro-oxidant enzyme p67(phox) subunit of nicotinamide adenine dinucleotide phosphate (NAD[P]H oxidase p67(phox)), an oxidative stress sensor was constructed, comprising the firefly luciferase (Fluc) reporter gene driven by the NAD(P)H p67(phox) promoter. MSCs cotransfected with NAD(P)H p67(phox)-Fluc and a cell viability reporter gene (cytomegalovirus-Renilla luciferase) were studied under in vitro and in vivo pro-oxidant conditions.
After in vitro validation of the sensor during low-serum culture, transfected MSCs were transplanted into a rat model of myocardial ischemia/reperfusion (IR) and monitored by using bioluminescence imaging. Compared with sham controls (no IR), cardiac Fluc intensity was significantly higher in IR rats (3.5-fold at 6 h, 2.6-fold at 24 h, 5.4-fold at 48 h; p < 0.01), indicating increased cellular oxidative stress. This finding was corroborated by ex vivo luminometry after correcting for Renilla luciferase activity as a measure of viable MSC number (Fluc:Renilla luciferase ratio 0.011 ± 0.003 for sham vs. 0.026 ± 0.004 for IR at 48 h; p < 0.05). Furthermore, in IR animals that received MSCs preconditioned with an antioxidant agent (tempol), Fluc signal was strongly attenuated, substantiating the specificity of the oxidative stress sensor.
Pathway-specific reporter gene imaging allows assessment of changes in the oxidative status of MSCs after delivery to ischemic myocardium, providing a template to monitor key biological interactions between transplanted cells and their host environment in living subjects.
本研究旨在验证一种可用于非侵入性成像祖细胞氧化状态的特定途径报告基因。
在细胞治疗研究中，报告基因成像在评估活体研究对象中细胞命运方面发挥了重要作用。在心肌损伤后，宿主组织中的有害刺激赋予移植细胞氧化应激，可能影响其存活和修复功能。
研究了大鼠间充质基质细胞（MSCs）在体外应激下氧化应激增加的表型证据。基于其上调烟酰胺腺嘌呤二核苷酸磷酸（NAD[P]H）氧化酶 p67（phox）亚单位的前氧化剂酶 p67（phox），构建了一种氧化应激传感器，由萤火虫荧光素酶（Fluc）报告基因驱动的 NAD（P）H p67（phox）启动子。在体外和体内前氧化剂条件下，研究了共转染 NAD（P）H p67（phox）-Fluc 和细胞活力报告基因（巨细胞病毒-Renilla 荧光素酶）的 MSCs。
在低血清培养过程中对传感器进行体外验证后，将转染的 MSCs 移植到大鼠心肌缺血/再灌注（IR）模型中，并通过生物发光成像进行监测。与假对照（无 IR）相比，IR 大鼠的心脏 Fluc 强度显着升高（6 h 时为 3.5 倍，24 h 时为 2.6 倍，48 h 时为 5.4 倍；p <0.01），表明细胞氧化应激增加。通过校正 Renilla 荧光素酶活性作为活 MSC 数量的指标（48 h 时 Fluc：Renilla 荧光素酶比值为 0.011 ± 0.003 用于 sham 与 0.026 ± 0.004 用于 IR；p <0.05），这一发现得到了体外发光计的证实。此外，在接受抗氧化剂（tempol）预处理的 MSCs 的 IR 动物中，Fluc 信号显着减弱，证实了氧化应激传感器的特异性。
特定途径报告基因成像可评估移植到缺血心肌后的 MSCs 氧化状态变化，为在活体研究对象中监测移植细胞与其宿主环境之间关键生物学相互作用提供了模板。
