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细胞凋亡形态学检测与观察

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hoechst33258染色,他们认为细胞发生凋亡时,染色质会固缩。 所以Hoechst染色时,细胞核会呈致密浓染,或呈碎块状致密浓染。
正常细胞核

刺激后有致密浓染的凋亡细胞
 

AO-EB染色法:
The image below shows human lymphoma cells treated with the chemotherapy agent camptothecin. The cells that are undergoing apoptosis appear yellow and show the characteristic membrane blebbing (bubble formation)seen in cells dying via apoptosis.

 

 

Promega公司的DeadEnd™比色法细胞凋亡检测系统能标记片断化的DNA,而这种片段化的DNA被视为是细胞凋亡的典型生化特征之一。DeadEnd™比色法细胞凋亡检测系统是一种在组织切片与培养细胞中标记凋亡细胞核的理想方法,与此同时,还可以做形态学上的评估。本文中,将展示此种方法在细胞凋亡的原位细胞, 动物模型及多种病理学组织切片中的应用。
实验流程:

 

结果:

抗Fas单克隆抗体(50ng/ml;克隆CH-11,Oncor)诱导的Jurkat细胞的细胞凋亡。DeadEnd™比色法细胞凋亡检测系统标记Fas单克隆抗体处理过的细胞核(16小时),而未处理过的则未被染色(右下角插图)。
 

凋亡的人类角化细胞被多聚甲醛固定,被荧光素标记的刀豆蛋白A染色。然后经丙酮渗透处理,被pi染色后能被共聚焦显微镜观察。本片显示的绿色荧光(外源性凝集素)勾勒出凋亡细胞表面的小泡,红色荧光(pi)被包被在其中。

10 µM喜树碱诱导的Jurkat cells(慢淋。G1检测点缺陷细胞) 凋亡。细胞然后经 Vybrant Apoptosis Assay Kit #4 (V13243).处理。凋亡细胞核被YO-PRO-1绿染,坏死细胞被pi红染。

Jurkat 人类白血病T细胞经1微升喜树碱处理,ps外翻,早期凋亡特异性的特征被annexin-5检测到。晚期凋亡和坏死细胞被pi染色。本图是由相应的荧光滤光片获取的。
 

HL-60 cells(急粒,p53缺陷)被喜树碱诱导3小时。DNA段端被荧光标记的脱氧核苷酸末端转移酶标记。

 

Detection of apoptosis in SK-N-MC neuroblastoma cells. Following a six-hour exposure to hydrogen peroxide, cells were labeled with Hoechst 33342 (H1399, H3570, H21492), tetramethylrhodamine ethyl ester (TMRE, T669) and rhodamine 110, bis-L-aspartic acid amide (R22122) for 15 minutes. Apoptotic cells show green cytosolic fluorescence resulting from cleavage of the rhodamine 110, bis-L-aspartic acid amide substrate by active caspase-3. The staining pattern of the Hoechst 33342 dye reveals that the majority of the rhodamine 110�positive cells also contain condensed or fragmented nuclei characteristic of apoptosis. Furthermore, the rhodamine 110�positive cells are also characterized by an absence of polarized mitochondria, as indicated by their failure to load the positively charged mitochondrial indicator TMRE. The image was contributed by A.K. Stout and J.T. Greenamyre, Emory University.

线虫发育中细胞的凋亡:
用共聚焦显微镜观察线虫胚胎,在正常胚胎CED-4位于线粒体。而凋亡细胞CED-4(红色)和核纤层蛋白(绿色)在核被膜(黄色)均可见。该实验有助于揭示,在细胞凋亡中CED-4从线粒体易位到核被膜
 

(1)形态学特征:发生时首先是染色质的凝集,嗜碱性染色增强,然后细胞核崩解此时线粒体保持形态正常。最后细胞体积缩小,一部分细胞质和核碎片进入由膜包被的程序死亡小体,他们从细胞表面出芽脱落,并被巨噬细胞、上皮细胞吞噬。
  (2)生化特征:染色质降解,核小体间连接DNA部位被降解,产生寡聚核小体DNA片段,即180-200DP整数倍的不同长度的DNA片断。
 

 

细胞形态学方面的资料:

1 光学显微镜和倒置显微镜
  (1) 未染色细胞:凋亡细胞的体积变小、变形,细胞膜完整但出现发泡现象,细胞凋亡晚期可见凋亡小体。贴壁细胞出现皱缩、变圆、脱落。
  (2) 染色细胞:常用姬姆萨染色、瑞氏染色等。凋亡细胞的染色质浓缩、边缘化,核膜裂解、染色质分割成块状和凋亡小体等典型的凋亡形态。
2 荧光显微镜和共聚焦激光扫描显微镜
  一般以细胞核染色质的形态学改变为指标来评判细胞凋亡的进展情况。
  常用的DNA特异性染料有:HO 33342 (Hoechst 33342),HO 33258 (Hoechst 33258), DAPI。三种种染料与DNA的结合是非嵌入式的,主要结合在DNA的A-T碱基区。紫外光激发时发射明亮的蓝色荧光。
  Hoechst是与DNA特异结合的活性染料,储存液用蒸馏水配成1mg/ml的浓度,使用时用PBS稀释,终浓度为10 ug/ml。
  DAPI为半通透性,用于常规固定细胞的染色。储存液用蒸馏水配成1mg/ml的浓度,使用终浓度一般为10 ug/ml。
  结果评判:细胞凋亡过程中细胞核染色质的形态学改变分为三期:Ⅰ期的细胞核呈波纹状(rippled)或呈折缝样(creased),部分染色质出现浓缩状态;Ⅱa期细胞核的染色质高度凝聚、边缘化;Ⅱb期的细胞核裂解为碎块,产生凋亡小体

不过我始终没有找到这种分类标准的文字依据。实际中也不好判定,各位战友有相关资料,热切盼望共享!

  3 透射电子显微镜观察
  结果评判:凋亡细胞体积变小,细胞质浓缩。凋亡Ⅰ期(pro-apoptosis nuclei)的细胞核内染色质高度盘绕,出现许多称为气穴现象(cavitations)的空泡结构(图2);Ⅱa期细胞核的染色质高度凝聚、边缘化;细胞凋亡的晚期,细胞核裂解为碎块,产生凋亡小体。
 

A comparison of normal and apoptotic cells.


confocal microscopy共聚焦显微镜用于检测凋亡

 

 

 

a microscope image of a group of normal cells and one apototic cell which binds annexin (green) on the surface. Annexin is labeled with fluorescein

In apoptosis condensation and fragmentation of chromatin occurs. Subsequently, nuclei loose their round or oval shape, bud and become fragmented - apoptotic bodies are formed

Nuclei of normal human fibroblasts in culture, in early spontaneous apoptosis
 

Nuclei of normal human fibroblasts in culture, in advanced spontaneous apoptosis

Apoptosis caused in HL60 cells by camptothecin (topoisomerase inhibitor). Images of nuclei in early apoptosis.

Apoptosis caused in HL60 cells by camptothecin (topoisomerase inhibitor). Images of nuclei in mid apoptosis

Apoptosis caused in HL60 cells by camptothecin (topoisomerase inhibitor). Images of nuclei in late apoptosis

A schematic view of a nucleus in a cell undergoing apoptosis: healthy cell (top, right), early apoptosis (top, left), advanced apoptosis (bottom, right), late apoptosis (bottom, left).

 

 

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