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首页> 外文期刊>Lab on a chip >Study of endothelial cell apoptosis using fluorescence resonance energy transfer (FRET) biosensor cell line with hemodynamic microfluidic chip system
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Study of endothelial cell apoptosis using fluorescence resonance energy transfer (FRET) biosensor cell line with hemodynamic microfluidic chip system

机译：带有血流动力学微流控芯片系统的荧光共振能量转移（FRET）生物传感器细胞系研究内皮细胞凋亡

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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.
机译：为了更好地了解在糖尿病条件下高血糖症如何诱发内皮细胞功能障碍，开发了一种血液动力学微流控芯片系统。该系统结合了基于caspase-3的荧光共振能量转移（FRET）生物传感器细胞系，通过使用可以模拟内皮细胞凋亡的微流控芯片，可以实时，后处理效果检测内皮细胞凋亡，并与有限的细胞样品结合使用。血管中的生理性脉动血流曲线。基于caspase-3的FRET生物传感器内皮细胞系（HUVEC-C3）可以产生基于FRET的传感器蛋白，能够探测caspase-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，从而感应凋亡。使用双标记荧光技术（yo pro-1和碘化丙啶）来验证基于FRET的胱天蛋白酶传感器揭示的发现。结果表明，在我们的血液动力学微流控芯片中应用高浓度葡萄糖并结合详尽的脉动血流曲线后，内皮细胞的凋亡和坏死率很高。两种凋亡检测技术（荧光方法和FRET生物传感器）具有可比性;但是FRET生物传感器具有更多优势，例如实时观察和便捷的操作过程，可以生成更准确和可靠的数据。此外，FRET生物传感器的激活还证实了异常搏动切应力引起的内皮细胞凋亡，而高浓度的葡萄糖则通过caspase-3途径。当内皮细胞在30达因cm〜（-2）的力竭力2 h下暴露于20 mM的高浓度葡萄糖时，观察到12％的凋亡率（与静态相比增加了4倍）。然后再施加10达15达因cm〜（-2）的正常脉动剪切应力。因此，本研究的最重要发现是开发一种结合了微流控芯片系统和FRET生物传感器的新型内皮细胞凋亡检测方法。这一发现可能为葡萄糖如何引起内皮细胞功能障碍提供新的见解，而内皮细胞功能障碍是糖尿病衍生并发症的主要原因。

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