用流式细胞术研究血液病细胞死亡生物学

Darzynkiewicz等提出的细胞死亡生物学,意在研究细胞死亡前、中、后的形态、生化和分子生物学等的全过程以及细胞死后邻近组织的反应。经过半个多世纪对细胞增殖动力学的研究后,人们终于认识到细胞死亡与增殖同等重要。两者的不平衡是多种疾病的根源,因而细胞死亡的研究成为近年来生命科学的研究热点。细胞凋亡在骨髓增生异常综合征,慢性淋巴细胞白血病,慢性粒细胞白血病,以及急性白血病发生发展中的作用已获得初步证实。深入研究其机理有利于发展新的靶向治疗。为了准确判断细胞死亡类型,可靠的实验技术至关重要。流式细胞术(FCM)能多参数、快速、定性、定量测定群体细胞中单个细胞的生物学过程,例如用光散射可测定细胞形态,用膜联蛋白(Annexin V),碘化丙啶(PI)和吖啶橙(AO)等,可测定早期细胞膜结构与通透功能。若丹明123(rhodamine 123)是线粒体特异标志。DNA经内切酶降解后在FCM-DNA图中出现亚G_1峰。用各种抗基因蛋白单克隆抗体如Bcl-2,Bcl-XL,Bax,ICE,c-myc,Fas/FasL等可测知单个细胞中与细胞凋亡相关基因蛋白表达及其间关系。Tunnel法与PI双标志法是判断凋亡细胞发生在哪个周期时相的最可靠方法。本文简要介绍各种方法的原理与质量控制以及如何正确判断实验结果。

Cell necrobiology study in hematological disorders by means of flow cytometry

The term cell necrobiology introduced by Darzynkiewicz et al comprises the life processes associated with morphological, biochemical, and molecular changes which predispose, precede and accompany celi death as well as the consequences and tissue response to celi death. Celi death has become a focus of interest for researchers from a variety disciplines since last decade. The cause of wide interest is based on the recognition that the imba-lance between the rate of cell growth and the loss of their ability to die on schedule play a main pathogenesis in certain diseases. For example, apoptosis has been proved to take a main part in pathogenesis of certain hemato-logical disorders, in myelodysplatic syndrome (MDS), chronic lymphocytic leukemia, chronic myelocytic leukemia and acute leukemias as well. Understanding of the basie mechanisms that underlie apoptosis will offer potential new targets for therapeutic treatment of the diseases. In order to conduct such research, techniques to reliably identify cell death modes are essential. Flow cytometry (FCM) offers all the advantages of rapid, multi-parameter analysis of large population of individual cells to investigate the biological processes associated with cell death. FCM is able to measure changes in cell morphology (by light scatter), plasma membrane structure and transport function(annexin V, propidium iodide, acridine orange, etc.), functions of cell organelles (rhodamine 123, etc.), endonucleolytic DNA degradation (sub-Gl peak) and apoptotic related gene expressions (Bcl-2, Bcl-XL, Bax, ICE, c-myc, Fas/FasL, etc. ). In situ labeling of DNA strand break by BrdUTP incorporation (Tunnel assay) and PI double staining offers a unique possibility to analyze the cell cycle position of apoptotic celis. The principles and quality control of the tests are introduced briefly. And improper use of FCM in analysis of cell death and in data interpretation are discussed.