细胞凋亡相关研究及检测技术 凋亡研究进展及检测.ppt

* * MitoGLO?, JC-1 Dye Optimized for Cell Culture. Mitochondrial depolarization can be detected by a unique fluorescent cationic dye, 5,5,6,6-tetrachloro-1,1,3,3-tetraethyl-benzamidazolocarbocyanin iodide, also known as JC-1 (reviewed in 6-8). The fluoroscence emission of JC-1 varies depending on the relative concentration of the dye, which in turn is determined by DY. JC-1 which can exist as a monomer or as aggregates, referred to as J-aggregates, excites at 488-490 nm. The monomeric form of the dye emits at 527 nm, whereas the J-aggregates emit at 590 nm. JC-1 dye has been used to monitor DY across the spectrum of species, from plant to worm to human.? Once inside the cell, the lipophilic MitoGLO?, bearing a delocalized positive charge rapidly enters the negatively charged mitochondria. As the concentration of MitoGLO? increases within polarized mitochondria, J-aggregates form which fluoresces red (590 nm). In cells containing mitochondria undergoing depolarization, MitoGLO? J-aggregates do not remain sequestered within the mitochondria and are distributed throughout the cytoplasm in its monomeric form. This event leads to a decrease in spectral shift and loss of red fluorescence. In some model systems, an increase in green fluorescence may also be observed during the cell death process. ? 6、细胞凋亡的分子水平检测方法 1）Caspase分子的检测 2）线粒体膜势能的检测 JC-1 3）凋亡相关分子的检测 促凋亡分子 抑凋亡分子 4）相关信号通路分子的检测 1）Caspase分子的检测 Caspase3、8和9，2，6等 Caspase检测方法 ①分光光度法检测 ②FACS检测 ③Western Blot检测 全长检测 剪切体检测 ①Caspase分光光度法试剂盒 检测原理：活化的Caspase与其特异性底物DEVD- pNA 结合切割，释放出游离pNA，通过测定其吸光度，根据其发光强度的高低代表其活化程度。 检测材料与方法 活细胞、新鲜或速深冷冻组织制成的细胞悬液——裂解——蛋白定量——加入底物反应——测定吸光度 ③Caspase 3 Western Blot检测 通过对Caspase底物PARP等的降解产物的检测反映Caspase的活性。 通过针对Caspase 催化的降解产物采用IHC、Western Blot、FCM进行检测。 原理： JC-1染料在正常细胞内聚集在线粒体内，形成多聚体，发红色荧光；而在凋亡细胞内，由于线粒体膜电位的破坏，不能聚集到线粒体内，以单体的形式存在于胞质内发绿色荧光。 2）线粒体膜势能（ JC-1 ）的检测 荧光显微镜检测 FACS检测 ②线粒体膜势能的流式检测(JC-1) 3）凋亡相关分子的检测 促凋亡分子 Bax, Bcl-Xs, Bak, Bad, Bid, AIF,Apaf-1和cytochrome c等 抑制凋亡分子 Bcl-2,