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  • 發布時間:2019-04-27 18:58 原文鏈接: 細胞凋亡形態學檢測與觀察

    hoechst33258染色,他們認為細胞發生凋亡時,染色質會固縮。 所以Hoechst染色時,細胞核會呈致密濃染,或呈碎塊狀致密濃染。
    正常細胞核

    刺激后有致密濃染的凋亡細胞 
     

    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?比色法細胞凋亡檢測系統能標記片斷化的DNA,而這種片段化的DNA被視為是細胞凋亡的典型生化特征之一。DeadEnd?比色法細胞凋亡檢測系統是一種在組織切片與培養細胞中標記凋亡細胞核的理想方法,與此同時,還可以做形態學上的評估。本文中,將展示此種方法在細胞凋亡的原位細胞, 動物模型及多種病理學組織切片中的應用。
    實驗流程:

     

    結果:

    抗Fas單克隆抗體(50ng/ml;克隆CH-11,Oncor)誘導的Jurkat細胞的細胞凋亡。DeadEnd?比色法細胞凋亡檢測系統標記Fas單克隆抗體處理過的細胞核(16小時),而未處理過的則未被染色(右下角插圖)。 
     

    凋亡的人類角化細胞被多聚甲醛固定,被熒光素標記的刀豆蛋白A染色。然后經丙酮滲透處理,被pi染色后能被共聚焦顯微鏡觀察。本片顯示的綠色熒光(外源性凝集素)勾勒出凋亡細胞表面的小泡,紅色熒光(pi)被包被在其中。 

    10 μM喜樹堿誘導的Jurkat cells(慢淋。G1檢測點缺陷細胞) 凋亡。細胞然后經 Vybrant Apoptosis Assay Kit #4 (V13243).處理。凋亡細胞核被YO-PRO-1綠染,壞死細胞被pi紅染。 

    Jurkat 人類白血病T細胞經1微升喜樹堿處理,ps外翻,早期凋亡特異性的特征被annexin-5檢測到。晚期凋亡和壞死細胞被pi染色。本圖是由相應的熒光濾光片獲取的。 
     

    HL-60 cells(急粒,p53缺陷)被喜樹堿誘導3小時。DNA段端被熒光標記的脫氧核苷酸末端轉移酶標記。 

     

    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.

    線蟲發育中細胞的凋亡:
    用共聚焦顯微鏡觀察線蟲胚胎,在正常胚胎CED-4位于線粒體。而凋亡細胞CED-4(紅色)和核纖層蛋白(綠色)在核被膜(黃色)均可見。該實驗有助于揭示,在細胞凋亡中CED-4從線粒體易位到核被膜 
     

    (1)形態學特征:發生時首先是染色質的凝集,嗜堿性染色增強,然后細胞核崩解此時線粒體保持形態正常。最后細胞體積縮小,一部分細胞質和核碎片進入由膜包被的程序死亡小體,他們從細胞表面出芽脫落,并被巨噬細胞、上皮細胞吞噬。
      (2)生化特征:染色質降解,核小體間連接DNA部位被降解,產生寡聚核小體DNA片段,即180-200DP整數倍的不同長度的DNA片斷。 
     

     

    細胞形態學方面的資料:

    1 光學顯微鏡和倒置顯微鏡
      (1) 未染色細胞:凋亡細胞的體積變小、變形,細胞膜完整但出現發泡現象,細胞凋亡晚期可見凋亡小體。貼壁細胞出現皺縮、變圓、脫落。
      (2) 染色細胞:常用姬姆薩染色、瑞氏染色等。凋亡細胞的染色質濃縮、邊緣化,核膜裂解、染色質分割成塊狀和凋亡小體等典型的凋亡形態。
    2 熒光顯微鏡和共聚焦激光掃描顯微鏡
      一般以細胞核染色質的形態學改變為指標來評判細胞凋亡的進展情況。
      常用的DNA特異性染料有:HO 33342 (Hoechst 33342),HO 33258 (Hoechst 33258), DAPI。三種種染料與DNA的結合是非嵌入式的,主要結合在DNA的A-T堿基區。紫外光激發時發射明亮的藍色熒光。
      Hoechst是與DNA特異結合的活性染料,儲存液用蒸餾水配成1mg/ml的濃度,使用時用PBS稀釋,終濃度為10 ug/ml。
      DAPI為半通透性,用于常規固定細胞的染色。儲存液用蒸餾水配成1mg/ml的濃度,使用終濃度一般為10 ug/ml。
      結果評判:細胞凋亡過程中細胞核染色質的形態學改變分為三期:Ⅰ期的細胞核呈波紋狀(rippled)或呈折縫樣(creased),部分染色質出現濃縮狀態;Ⅱa期細胞核的染色質高度凝聚、邊緣化;Ⅱb期的細胞核裂解為碎塊,產生凋亡小體

    不過我始終沒有找到這種分類標準的文字依據。實際中也不好判定,各位戰友有相關資料,熱切盼望共享!

      3 透射電子顯微鏡觀察
      結果評判:凋亡細胞體積變小,細胞質濃縮。凋亡Ⅰ期(pro-apoptosis nuclei)的細胞核內染色質高度盤繞,出現許多稱為氣穴現象(cavitations)的空泡結構(圖2);Ⅱa期細胞核的染色質高度凝聚、邊緣化;細胞凋亡的晚期,細胞核裂解為碎塊,產生凋亡小體。 
     

    A comparison of normal and apoptotic cells. 


    confocal microscopy共聚焦顯微鏡用于檢測凋亡

     

     

    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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