秀丽隐杆线虫和芽殖酵母中乏氧诱至的假死状态之细胞机制研究

Suspended animation, a totally reversible hypometabolic phenomenon characterized by the complete arrest of biological processes, such as cell division, development and movement, can be induced by unfavorable conditions such as anoxia, extremely low oxygen tension (< 2 ppm). It is an adaptive process observed in many organisms from single cell to multicellular organisms and from invertebrates to vertebrates, implying the high degree of evolutionary conservation. Total cellular arrest has been proved to be a clinical valuable technique especially in buying time for providing critical care and surgeries. Though various organisms including mammals have been shown to be capable of entering suspended animation under anoxia, the cellular mechanism of oxygen sensing and cell cycle arrest are still largely unknown. Therefore, the aim of this study is to use C. elegans and S. cerevisiae to investigate their phenotypes and behaviors upon anoxic exposure and elucidate the cellular mechanism of anoxia-induced suspended animation. By using an anoxic incubation chamber and a novel microfluidic chip which is consisted of an incubation unit, a microdissection unit and an embryo capture array, we were able to strictly control the oxygen level, complying the anoxic exposure requirement (< 2 ppm). Our results demonstrated that after 24 h of anoxic exposure, around 90% of C. elegans at various life cycle stages could recover from suspended animation in less than 20 min in normoxia. Brief exposure to anoxia (< 8 h) does not have significant harmful effects on the reproductive potential, embryonic viability and the movement of C. elegans. On the other hand, S. cerevisiae can tolerate anoxic stress for a longer exposure (9 days). Furthermore, yeast cells arrested at G1/S, S or G2/M phase showed similar abilities to enter suspended animation, implying that across the cell cycle, there is coordination of sensing oxygen and arresting the cell cycle to enter suspended animation. In addition, our systematic bioinformatic analysis identified a number of genes in C. elegans, yeast and human, which may be required for anoxia-correlated phenotypes, by interacting with chromatin remodelers, histones modifiers and cell cycle checkpoint proteins. These results indicated that both S. cerevisiae and C. elegans, although of different evolutionary complexity, showed similar abilities to tolerate anoxia. The current study would allow the identification of novel anoxia-specific sensing proteins and reveal the underlying mechanisms of cell cycle arrest, maintenance and recovery.

假死状态是一种可逆转的低代谢状态，其主要特征为暂停生物过程，这状态能由乏氧（含氧<2ppm）所导致。虽然不同的生物已显示能够在乏氧下进入假死状态，但是其中的氧感应和细胞活动停滞机制仍然是未知的。因此，本研究旨在使用乏氧培养箱、新型微流体芯片及活体成像技术以研究秀丽隐杆线虫和芽殖酵母在乏氧下的表型和行为，以阐明乏氧诱至假死状态背后的细胞机制。在24小时乏氧暴露后，约90％在各种生长阶段的线虫可以在不到20分钟内在常氧环境中恢复活动。短时间乏氧暴露对线虫的生殖力、胚胎生存率和运动能力没有显著的影响。我们的生物信息学分析亦预测了超过100个于线虫、酵母和人类中与乏氧表型相关的保守基因。 这些工作基础表明，线虫和酵母虽然具有不同的进化复杂性，但都显示出类似的耐受乏氧能力。此研究将鉴定特别为感应乏氧特异性的新型蛋白，并揭示细胞于乏氧环境中，维持细胞周期停顿、和于重返常氧环境时恢复细胞活动的基础机制。

假死是一种细胞保护机制，在各种不利的环境条件下，如热、冷、干旱和无氧条件下，暂时停止所有细胞活动，包括细胞周期进程、代谢和运动。当环境压力被消除后，生物体可以从假死中可逆地恢复，不会造成任何伤害或不良影响。虽然这种现象可能不是每天都能观察到，但这是一种吸引人的、保守的机制，在许多不同的生物上均可观察到，如模式生物斑马鱼，线虫和酵母。..当线虫暴露在极低的氧气水平(小于0.01%)下时，会发生无氧诱导的假死，而低氧诱导因子(HIF)并不是无氧生存所必需的。到目前为止，已知的线虫无氧生存所需的细胞周期调节因子只有纺锤体装配检查点蛋白MAD-3、2和BUB-3，以及芽殖酵母线粒体逆行信号途径。然而，还没有系统的、无偏倚的筛选在任何完整的有机体模型中进行，以进一步确定与无氧生存有关的新基因和途径。. .在前期工作中，我们研究了无氧对线虫不同生长阶段的生存、行为和生殖表型的影响。在不同的细胞阶段，芽殖酵母可以进入假死状态并在无氧条件下存活。.此外，我们利用芽殖酵母非必需基因缺失库(含4773个突变体)，对无氧恢复后生长不良的突变体进行了全基因组筛选。我们已经鉴定了34个无氧诱导休眠的(AIS)基因，它们或具有未知的功能，或在细胞周期调控、线粒体、细胞内运输中发挥作用。其中70%在人类中具有保守的同源基因。..阐明感知氧的分子机制和途径，细胞周期活动的停滞机制和如何从无氧中恢复是我们理解细胞稳态、生物-环境相互作用和生命进化的基础。从酵母细胞中获得的基本知识可用于优化紧急保存和复苏(EPR)方案，以诱导更长时间和无害的假死。这样的应用将有助于延长病人的手术时间，延长捐赠的细胞、组织或器官移植的保存时间。因此，我们目前对缺氧诱导的假死和恢复的研究不仅与细胞和分子生物学、遗传学、进化生物学有关，而且具有医学和临床意义。