Ben May Department for Cancer Research, Gordon Center for Integrative Sciences, University of Chicago, IL, USA.
J Pathol. 2010 Jun;221(2):117-24. doi: 10.1002/path.2694.
Autophagy is a fundamental and phylogenetically conserved self-degradation process that is characterized by the formation of double-layered vesicles (autophagosomes) around intracellular cargo for delivery to lysosomes and proteolytic degradation. The increasing significance attached to autophagy in development and disease in higher eukaryotes has placed greater importance on the validation of reliable, meaningful and quantitative assays to monitor autophagy in live cells and in vivo in the animal. To date, the detection of processed LC3B-II by western blot or fluorescence studies, together with electron microscopy for autophagosome formation, have been the mainstays for autophagy detection. However, LC3 expression levels can vary markedly between different cell types and in response to different stresses, and there is also concern that over-expression of tagged versions of LC3 to facilitate imaging and detection of autophagy interferes with the process itself. In addition, the realization that it is not sufficient to monitor static levels of autophagy but to measure 'autophagic flux' has driven the development of new or modified approaches to detecting autophagy. Here, we present a critical overview of current methodologies to measure autophagy in cells and in animals.
自噬是一种基本的、进化上保守的自我降解过程，其特征是双层囊泡（自噬体）在细胞内货物周围形成，以便递送至溶酶体进行蛋白水解降解。自噬在高等真核生物的发育和疾病中的重要性不断增加，这使得对可靠、有意义和定量的检测方法的验证变得更加重要，以便在活细胞和动物体内监测自噬。迄今为止，通过 Western blot 或荧光研究检测加工后的 LC3B-II，以及电子显微镜检测自噬体的形成，一直是自噬检测的主要方法。然而，LC3 的表达水平在不同细胞类型之间差异很大，并且对不同的应激反应也有很大的影响，此外，还存在一种担忧，即过表达标记的 LC3 版本以促进自噬的成像和检测会干扰该过程本身。此外，人们认识到，仅仅监测自噬的静态水平是不够的，还需要测量“自噬通量”，这推动了新的或改进的检测自噬方法的发展。在这里，我们对目前用于检测细胞和动物中自噬的方法进行了批判性的综述。
