School of Electrical and Electronic Engineering, Nanyang Technological University, 50 Nanyang Avenue, Singapore 639798.
Lab Chip. 2013 Jul 21;13(14):2693-700. doi: 10.1039/c3lc50105a.
To better understand how hyperglycemia induces endothelial cell dysfunction under the diabetic conditions, a hemodynamic microfluidic chip system was developed. The system combines a caspase-3-based fluorescence resonance energy transfer (FRET) biosensor cell line which can detect endothelial cell apoptosis in real-time, post-treatment effect and with a limited cell sample, by using a microfluidic chip which can mimic the physiological pulsatile flow profile in the blood vessel. The caspase-3-based FRET biosensor endothelial cell line (HUVEC-C3) can produce a FRET-based sensor protein capable of probing caspase-3 activation. When the endothelial cells undergo apoptosis, the color of the sensor cells changes from green to blue, thus sensing apoptosis. A double-labeling fluorescent technique (yo pro-1 and propidium iodide) was used to validate the findings revealed by the FRET-based caspase sensor. The results show high rates of apoptosis and necrosis of endothelial cells when high glucose concentration was applied in our hemodynamic microfluidic chip combined with an exhaustive pulsatile flow profile. The two apoptosis detection techniques (fluorescent method and FRET biosensor) are comparable; but FRET biosensor offers more advantages such as real-time observation and a convenient operating process to generate more accurate and reliable data. Furthermore, the activation of the FRET biosensor also confirms the endothelial cell apoptosis induced by the abnormal pulsatile shear stress and high glucose concentration is through caspase-3 pathway. A 12% apoptotic rate (nearly a 4-fold increase compared to the static condition) was observed when the endothelial cells were exposed to a high glucose concentration of 20 mM under 2 h exhaustive pulsatile shear stress of 30 dyne cm(-2) and followed with another 10 h normal pulsatile shear stress of 15 dyne cm(-2). Therefore, the most important finding of this study is to develop a novel endothelial cell apoptosis detection method, which combines the microfluidic chip system and FRET biosensor. This finding may provide new insight into how glucose causes endothelial cell dysfunction, which is the major cause of diabetes-derived complications.
为了更好地理解高血糖在糖尿病条件下如何诱导内皮细胞功能障碍，开发了一种血流动力学微流控芯片系统。该系统结合了基于半胱天冬酶-3 的荧光共振能量转移 (FRET) 生物传感器细胞系，该细胞系可以实时检测内皮细胞凋亡，进行治疗后效果评估，并仅使用微流控芯片即可对有限的细胞样本进行分析，该芯片可模拟血管中的生理脉动流谱。基于半胱天冬酶-3 的 FRET 生物传感器内皮细胞系 (HUVEC-C3) 可产生一种基于 FRET 的传感器蛋白，能够探测半胱天冬酶-3 的激活。当内皮细胞发生凋亡时，传感器细胞的颜色从绿色变为蓝色，从而感知凋亡。使用双标记荧光技术（yo pro-1 和碘化丙啶）验证了基于 FRET 的半胱天冬酶传感器检测结果。结果表明，当高葡萄糖浓度与耗尽的脉动流谱结合应用于我们的血流动力学微流控芯片时，内皮细胞的凋亡和坏死率很高。两种凋亡检测技术（荧光法和 FRET 生物传感器）具有可比性；但 FRET 生物传感器具有更多优势，例如实时观察和方便的操作过程，可生成更准确可靠的数据。此外，FRET 生物传感器的激活还证实了异常脉动剪切力和高葡萄糖浓度诱导的内皮细胞凋亡是通过半胱天冬酶-3 途径发生的。当内皮细胞在 20 mM 高葡萄糖浓度下暴露于 30 dyne cm(-2) 2 小时耗尽的脉动剪切力后，再用 15 dyne cm(-2) 正常脉动剪切力持续 10 小时，凋亡率达到 12%（与静态条件相比增加了近 4 倍）。因此，本研究的最重要发现是开发了一种新的内皮细胞凋亡检测方法，该方法结合了微流控芯片系统和 FRET 生物传感器。这一发现可能为葡萄糖引起内皮细胞功能障碍的机制提供新的见解，而内皮细胞功能障碍是糖尿病衍生并发症的主要原因。
