Hirano Yu, Nishimiya Yoshiyuki, Kowata Keiko, Mizutani Fumio, Tsuda Sakae, Komatsu Yasuo
Research Institute of Genome-based Biofactory, National Institute of Advanced Industrial Science and Technology (AIST), 2-17-2-1 Tsukisamu-Higashi, Toyohira, Sapporo, Japan.
Anal Chem. 2008 Dec 1;80(23):9349-54. doi: 10.1021/ac8018334.
Antifreeze proteins (AFPs) can protect cells from hypothermic damage; however, their mechanism of action remains unclear. Scanning electrochemical microscopy (SECM) can evaluate the size and activities of cells, although long-term continuous monitoring has been unsuccessful. We constructed a novel, fully automated, time-lapse SECM system and investigated the cell preservation effect of AFPs by analyzing single cellular topography at low temperatures. From the SECM measurements, mammalian cells (HepG2), treated in Euro-Collins (EC) solution at 4 degrees C, began to swell at 8 h and then immediately ruptured. In AFP-containing EC solution, the cellular size did not change until 16 h and then gradually increased and finally ruptured. In addition, the cellular height at rupture point significantly increased in the presence of AFPs. These results suggest that AFPs stabilize the cellular membrane and protect cells from hypothermic damage. This SECM system allowed us to observe the single cellular response to hypothermia by long-term automatic scanning and will be applicable for analysis to other cellular activities and topographies.
抗冻蛋白（AFPs）可以保护细胞免受低温损伤；然而，其作用机制仍不清楚。扫描电化学显微镜（SECM）可以评估细胞的大小和活性，尽管长期连续监测尚未成功。我们构建了一种新型的、全自动的、延时SECM系统，并通过分析低温下的单细胞形貌来研究AFPs对细胞的保存效果。根据SECM测量结果，在4℃的Euro-Collins（EC）溶液中处理的哺乳动物细胞（HepG2）在8小时时开始肿胀，然后立即破裂。在含有AFPs的EC溶液中，细胞大小直到16小时才发生变化，然后逐渐增大，最终破裂。此外，在AFPs存在的情况下，破裂点处的细胞高度显著增加。这些结果表明，AFPs可稳定细胞膜并保护细胞免受低温损伤。该SECM系统使我们能够通过长期自动扫描观察单细胞对低温的反应，并将适用于分析其他细胞活动和形貌。
