Jemaà Mohamed, Fezai Myriam, Bissinger Rosi, Lang Florian
Department of Internal Medicine III, University of Tuebingen, Tuebingen, Germany.
Department of Laboratory Medicine, Translational Cancer Research, Lund University, Lund, Sweden.
Cell Physiol Biochem. 2017;43(2):431-444. doi: 10.1159/000480469. Epub 2017 Sep 1.
Suicidal erythrocyte death or eryptosis contributes to or even accounts for anemia in a wide variety of clinical conditions, such as iron deficiency, dehydration, hyperphosphatemia, vitamin D excess, chronic kidney disease (CKD), hemolytic-uremic syndrome, diabetes, hepatic failure, malignancy, arteriitis, sepsis, fever, malaria, sickle-cell disease, beta-thalassemia, Hb-C and G6PD-deficiency, Wilsons disease, as well as advanced age. Moreover, eryptosis is triggered by a myriad of xenobiotics and endogenous substances including cytotoxic drugs and uremic toxins. Eryptosis is characterized by cell membrane scrambling with phosphatidylserine exposure to the erythrocyte surface. Triggers of eryptosis include oxidative stress, hyperosmotic shock, and energy depletion. Signalling involved in the regulation of eryptosis includes Ca2+ entry, ceramide, caspases, calpain, p38 kinase, protein kinase C, Janus-activated kinase 3, casein kinase 1α, cyclin-dependent kinase 4, AMP-activated kinase, p21-activated kinase 2, cGMP-dependent protein kinase, mitogen- and stress-activated kinase MSK1/2, and ill-defined tyrosine kinases. Inhibitors of eryptosis may prevent anaemia in clinical conditions associated with enhanced eryptosis and stimulators of eryptosis may favourably influence the clinical course of malaria. Additional experimentation is required to uncover further clinical conditions with enhanced eryptosis, as well as further signalling pathways, further stimulators, and further inhibitors of eryptosis. Thus, a detailed description of the methods employed in the analysis of eryptosis may help those, who enter this exciting research area. The present synopsis describes the experimental procedures required for the analysis of phosphatidylserine exposure at the cell surface with annexin-V, cell volume with forward scatter, cytosolic Ca2+ activity ([Ca2+]i) with Fluo3, oxidative stress with 2',7'-dichlorodihydrofuorescein diacetate (DCFDA), glutathione (GSH) with mercury orange 1(4-chloromercuryphenyl-azo-2-naphthol), lipid peroxidation with BODIPY 581/591 C11 fluorescence, and ceramide abundance with specific antibodies. The contribution of kinases and caspases is defined with the use of the respective inhibitors. It is hoped that the present detailed description of materials and methods required for the analysis of eryptosis encourages further scientists to enter this highly relevant research area.
自杀性红细胞死亡或红细胞凋亡在多种临床病症中会导致甚至引发贫血，如缺铁、脱水、高磷血症、维生素D过量、慢性肾病（CKD）、溶血尿毒综合征、糖尿病、肝衰竭、恶性肿瘤、动脉炎、败血症、发热、疟疾、镰状细胞病、β地中海贫血、Hb-C和葡萄糖-6-磷酸脱氢酶（G6PD）缺乏症、威尔逊氏病以及高龄。此外，红细胞凋亡由多种外源性物质和内源性物质引发，包括细胞毒性药物和尿毒症毒素。红细胞凋亡的特征是细胞膜磷脂酰丝氨酸外翻暴露于红细胞表面。红细胞凋亡的触发因素包括氧化应激、高渗休克和能量耗竭。参与红细胞凋亡调节的信号传导包括钙离子内流、神经酰胺、半胱天冬酶、钙蛋白酶、p38激酶、蛋白激酶C、Janus激活激酶3、酪蛋白激酶1α、细胞周期蛋白依赖性激酶4、AMP激活激酶、p21激活激酶2、环鸟苷酸依赖性蛋白激酶、丝裂原和应激激活激酶MSK1/2以及未明确的酪氨酸激酶。红细胞凋亡抑制剂可能预防与红细胞凋亡增强相关的临床病症中的贫血，而红细胞凋亡刺激剂可能对疟疾的临床病程产生有利影响。需要进一步实验以发现更多红细胞凋亡增强的临床病症以及更多红细胞凋亡的信号传导途径、刺激剂和抑制剂。因此，对红细胞凋亡分析中所采用方法的详细描述可能会帮助那些进入这一令人兴奋的研究领域的人。本综述描述了用膜联蛋白-V分析细胞表面磷脂酰丝氨酸暴露、用前向散射分析细胞体积、用Fluo3分析胞质钙离子活性（[Ca2+]i）、用2',7'-二氯二氢荧光素二乙酸酯（DCFDA）分析氧化应激、用汞橙1（4-氯汞苯基偶氮-2-萘酚）分析谷胱甘肽（GSH）、用BODIPY 581/591 C11荧光分析脂质过氧化以及用特异性抗体分析神经酰胺丰度所需的实验程序。激酶和半胱天冬酶的作用通过使用各自的抑制剂来确定。希望本对红细胞凋亡分析所需材料和方法的详细描述能鼓励更多科学家进入这一高度相关的研究领域。
