细胞生物学第十三章 程序性细胞死亡与细胞衰老

1、CELL BIOLOGY Chapter 13 Programmed Cell Death and Cell Ageing Section 1 Programmed Cell Death Programmed Cell Death Apoptosis 细胞凋亡 Three ways of Necrosis 细胞坏死 animal cell death Autophagy 细胞自噬 Programmed Cell Death Apoptosis History Kerr observance 1965 Definition in 1972 Conception apoptosis apo ptosis 源自古希腊语 falling off 意 指花瓣或树叶的脱落 凋零 Interchangeably with programmed cell death The cells activate an intracellular death program and kill themselves in a controlled way Programmed Cell Death Morphol

2、ogical features during the programmed cell death 1 Initiation the disappearance of the cell surface structure such as microvilli the loss of cell contaction the mild convolution of the ER with plasm the cytoskeleton collapse the disassembles of the nuclear envelope and chromatin compaction and segregation Programmed Cell Death Morphological features during the programmed cell death 2 Formation of the apoptotic bodies nuclear chromatin break up into fragments the cell surface blebs and the cell b

3、reaks up into membrane enclosed fragments called apoptotic bodies Programmed Cell Death Morphological features during the programmed cell death 3 Engulf owing to the loss of contaction with the neighboring cell the apoptotic bodies will quickly be engulfed by neighboring cell or macrophages before their content to be spilled out Programmed Cell Death The typical feature of the programmed cell death The neat death without content spilling No damaging inflammatory response ATP depending cell self

4、killing Owing to the above features the apoptosis has been overlooked for a long time Programmed Cell Death Biological significance of cell apoptosis During animal development programmed cell death eliminates no longer needed structure For example When a tadpole changes into a frog at metamorphosis the cell in the tail die and the tail which is not needed in the frog disappears Programmed Cell Death Biological significance of cell apoptosis During animal development programmed cell death elimina

5、tes unwanted cells for example helps sculpt hands and feet during embryonic development Programmed Cell Death Biological significance of cell apoptosis In many other cases cell death helps regulate cell numbers In the developing nervous system for example cell death adjusts the number of nerve cells to match the number of target cells that the nerve cells connect to Programmed Cell Death Biological significance of cell apoptosis Programmed cell death also functions as a quality control process E

6、liminating cells that are abnormal misplaced nonfunctional or potentially dangerous to the animal Example 1 淋巴细胞的克隆选择过 程中 凡是不能区分 自我 与 异我 抗原的淋巴细 胞均发生凋亡 由此产生了机体的免疫耐受 否则会发生自身免疫疾病 Example 2 在成熟个体中 机体通过调节细 胞凋亡和细胞增殖的速度来维持组织器 官数量稳定 if a rat is treated with the drug Phenobarbital which stimulates liver cell division and thereby liver enlargement and then the Phenobarbital treatment is stopped apoptosis in the liver greatly increases until the liver has returned to its original size usually within a week

7、 or so Thus the liver is kept at a constant size through the regulation of both the cell death rate and the cell birth rate although the control mechanisms responsible for such regulation are largely unknown Programmed Cell Death Apoptosis related disease Decreased apoptosis 例如 自身免疫性淋巴增生综合症 ALPS 患者体内的Fas配体 信号 和Fas蛋白 受体 均 发生突变 使得增生的T 淋巴细胞无法正常凋亡 Excessive apoptosis 导致组织及免疫功能的丧失 或引发 自身免疫疾病和炎症 例如爱滋病 AIDS 患者表现为过 多 的淋巴 非淋巴细胞凋亡 免疫力低下 Programmed Cell Death Detection of cell apoptosis I Morphological obs

8、ervance Staining 台酚兰 trypan blue 活细胞排斥 但可使死细胞着色 DAPI 是常用的一种与DNA结合的荧光染料 用DAPI染色借 助荧光显微镜 可以观察到细胞核的形态变化 姬姆萨 Giemsa 可以使染色质着色 便于在普通光学显 微镜下观察到染色质固缩 凋亡小体的形成等凋亡过程 透射和扫描电镜 可观察凋亡细胞的形态 结构变化 如染色 质固缩 凋亡小体的形成 细胞发泡等现象 Programmed Cell Death A Normal cellB Apoptosis Apoptotic bodies 正常 a 和调亡 b 的T细胞杂交瘤细胞的扫瞄电镜照片 Programmed Cell Death Detection of cell apoptosis II DNA electrophorsis Endonuclease cleaves the chromosomal DNA into fragments of distinctive size because the cleavages occur in the linker regions between

9、 nucleosomes the fragments separate into a characteristic ladder pattern when analyzed by gel electrophoresis Programmed Cell Death Detection of cell apoptosis Lane 1 0 h 2 1 h 2 0kbp 3 2 h 4 3 h 5 4 h 6 Control 7 Marker 1 0 0 5 0 2 180 200 bp DNA ladder can be detected Detection of cell apoptosis 180 200 bp DNA ladder can be detected Programmed Cell Death Detection of cell apoptosis TUNEL测定法 DNA断裂 的原位末端标记法 针对DNA分子中断裂缺口中的3 OH进行原位标记 凋亡细胞的核 DNA中产生的3 OH末端 可藉助 荧光素进行原位标记 并用荧光显 微镜进行观察 Programmed Cell

10、Death 细胞凋亡的检测 方法 慧星电泳法 慧星电泳法 comet assay 其原理是将单个细胞悬浮于琼脂 糖凝胶中 经裂解处理后 再在电场中进行短时间的电泳 并用 荧光染料染色 凋亡细胞中的DNA降解片段 在电场中泳动速度 较快 使细胞核呈现出一种慧星式的图案 而正常的无DNA断裂 的核在泳动时保持圆球形 这是一种快速简便的凋亡检测法 Programmed Cell Death 细胞凋亡的检测 方法 流式细胞分析 与正常完整的二倍体细胞相比 凋亡细胞DNA发生 断裂和丢失 呈亚二倍体状态 采用碘化丙啶染色 使DNA产生激发荧光 流式细胞仪能够检测 出凋亡 的亚二倍体细胞 Programmed Cell Death Molecular Mechanism of Apoptosis Early researches MIT Robert Horvitz 1986 C elegans a millimeter long transparent body only a few cell types from zygote to mature adult only in 3 5days S

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