细胞生物学课件：第14章 细胞衰老与死亡癌变

1、CHAPTER 14细胞衰老、死亡与癌变 Cell aging, Death and Cancer 1OUTLINE14.1 细胞衰老 (Cell Aging)14.1.1 衰老的概念14.1.2 细胞的寿限14.1.3 细胞衰老的特征14.1.4 细胞衰老的理论14.2 细胞死亡 (Cell Death)14.2.1 程序性细胞死亡的基本特性14.2.2 程序性细胞死亡的机理14.2.3 程序性细胞死亡的生物学意义14.3 癌细胞 （Cancer）14.3.1 癌生物学14.3.2 癌的起因：物理和化学致癌物14.3.3 肿瘤发生的遗传学2314.1 细胞衰老14.1.1 衰老的概念衰老(s

2、enescing，aging)是机体在退化时期生理功能下降和紊乱的综合表现,是不可逆的生命过程。细胞的衰老与死亡是新陈代谢的自然现象。机体的衰老与细胞的衰老相关联。 现代人类面临着3种衰老：生理性衰老病理性衰老心理性衰老 414.1.2 细胞的寿限：1961年，Hayflick 首次报道了体外培养的人成纤维细胞具有增殖分裂的极限,且分裂能力与个体的年龄有关。来源于胚胎 分裂传代50次后开始死亡来源于成年组织 培养1530代就开始死亡动物体细胞在体外可传代的次数，与物种的寿命有关。5体外培养的年轻和老的人成纤维细胞的显微形态6物种的寿命与体外培养时细胞传代次数的关系长寿物种的细胞体外培养的代数比

3、短寿物种的细胞代数多7 Hayflick界限 (Hayflick life span): 细胞至少是体外培养细胞的增殖能力不是无限的，而是有一定界限，细胞的衰老控制着细胞的分裂次数, 进而控制着细胞的数量。细胞的寿限：各类细胞本身的寿命差别显著, 一般说来, 能够保持持续分裂能力的细胞是不容易衰老的。分化程度高又不分裂的细胞寿命是有限的。814.1.3 细胞衰老的特征细胞内水分减少色素生成和色素颗粒沉积衰老过程中细胞质膜的变化：流动性降低；兴奋性降低；配体受体复合物形成下降衰老过程中线粒体的变化数量减少，体积变大，膜破坏，DNA突变细胞核的变化核膜内折；染色质固缩；端粒缩短细胞骨架的变化蛋白质

4、合成的变化合成速度降低，蛋白定位改变2022/9/299MorphologySenescent cells become flattened, enlarged and have increased -galactosidase ( -半乳糖苷酶) activityIncreased size of nucleus and nucleoliIncreased number of multinucleated cellsIncreased number of lysosomes, Golgi and cytoplasmic microfilaments2022/9/2910Senescent c

5、ells undergo three phenotypic changes1114.1.4 细胞衰老的理论细胞衰老的线粒体损伤论 (Mitochondria ageing)自由基理论：氧自由基 (Reactive oxygen species (ROS)细胞衰老的端粒假说 (Replicative senescence and telomere shortening)细胞衰老的表观调控: (Epigenetic regulation of senescence) 衰老的进化论衰老的突变积聚、互逆多效理论12自由基攻击细胞的证据2022/9/2913Replicative senescence

6、is the progressive shortening of telomeres at chromosome endsCritically short telomeres trigger activation of cell cycle checkpointsPermanent cell cycle growth arrest due to activated cell cycle checkpoints, similar to those activated upon double strand breakageCells metabolically active but cannot

7、continue to divide, unlike quiesence 静止Replicative senescence2022/9/2914In the early embryonic period, cells have a determined length of telomere endings. As organism develops by cell differentiation, cells keep proliferating and during each division, telomeres get shorter because of replication mec

8、hanism specificity. So nature determined that as organism get older, telomeres get shorter and cell goes to death. 2022/9/2915Telomere dysfunction contributes to cancer X-rays UV OthersOxidative stressOxidative stressTelomeredysfunctionDNA damageCheckpoint activationSenescenceor apoptosisAccumulatio

9、n During lifeInactivatingmutationsp19ARFp53Stem CellCancerSenescence2022/9/2916Epigenetic Regulation of Senescence Epigenetics entails the study of the switching on and off of genes during development, cell proliferation, senescence and also by environmental insults. Genome modifications resulting f

10、rom epigenetic changes appear to play a critical role in the cellular senescene. Scatter experimental evidence suggests that epigenetic changes could also be critical determinants of cellular senescence and organisms senescence. 2022/9/2917 Histone deacetylases (HDACs) participate in senescence Elev

11、ated HDAC activity appears causally related to cellular senescence, as overexpression of a p300 mutant protein, or treatment with a specific chemical inhibitor of p300, results in irreversible growth arrest and senescence of normal human cells. DNA methylationSequential loss of DNA methylation could

12、 act as an alternative counting mechanism. A progressive loss of 5-methylcytosine in genomic DNA occurs during serial passage of normal cells in culture. The extent of CpG methylation also decreases during aging of organisms. On the other hand, immortal cell lines maintain constant levels of DNA met

13、hylation.2022/9/2918Chromatin remodeling and senescenceGenes in the p53, Rb, and ING (inhibitor of growth) pathways affect cell senescence and are capable of regulating gene expression through chromatin remodeling.p16INK4a is required for hSNF5 chromatin-remodeler induced cellular senescence in mali

14、gnant tumor cells PASG, an SNF2 family member, is essential for properly maintaining normal DNA methylation and gene expression patterns. Disruption of PASG leads to accumulation of senescence-associated tumor suppressor genes, and increased senescence- associated galactosidase as well as age-relate

15、d phenotypes.2022/9/2919Modification of different amino acid residues in histone H3 leads either to activation or repression of transcription.2022/9/2920RNA Degradation and Aging Model of age-related changes in AU-rich elements (ARE)-directed mRNA decay. As cells age, HuR levels decline, shifting th

16、e balance to mRNA degradation. Many ARE-mRNAs encoding proteins that contribute to proliferation, thus, decline which contributes to the phenotype of senescence. 2022/9/2921The role of genetics in determining life-span is complex and paradoxical. Although the heritability of life-span is relatively

17、minor, some genetic variants significantly modify senescence of mammals and invertebrates, with both positive and negative impacts on age-related disorders and life-spans. The Role of Genetics in Senescence It appears certain that DNA mutations and chromosomal abnormalities increase with age in mice

18、 and humans 2022/9/2922A mutant model mouse is useful for studies of aging. The klotho phenotype (premature aging) is caused by a disruption of the single gene, klotho. 2022/9/2923Cellular senescence involved in genetic errorsThere is an invariant relationship between life span and the number of ran

19、dom mutations. A number of studies at a number of gene loci have shown that somatic mutations of a variety of types accumulate with age. Deficient in DNA Repair and Transcription induce Premature Aging in Mice.TTD and XPD, genes for DNA repair and replication. SCIENCE VOL 296 17 MAY 2002Photograph o

20、f a 3-week-old XPA/TTD double-mutant (left), TTD (middle), and XPA (right) mouse.2022/9/2924Evolutionary Theory of SenescenceAging is a by-product of natural selection due to lack of selective pressure for the post-reproductive individual. Any individual has a probability to reproduce. It is zero at

21、 birth and reaches a peak in young adults. Then, it decreases due to the increased probability of death linked to various external (predators, illnesses, accidents) and internal causes (aging).25早老症儿童26细胞衰老的分子途径p19ARF/p53/p21Cip1 : p16INK4a/Rb端粒-p53-PGCCDK inhibitorsSenescence signal27Role of p53 in

22、 G1 arrest induced by DNA damage Induction of p21 via p53 activation p21: Cdk inhibitor. inhibit DNA synthesis by interacting with PCNA (a subunit of DNA polymerase ) 28Rb蛋白对细胞周期的调节p16INK4a2022/9/2929 Polycomb group protein BMI1 has been linked to proliferation, senescence and apoptosis.30Telomere-p

23、53-PGC PGC (perioxisome proliferator-activated receptor gamma coactivator)细胞代谢及线粒体功能的主要调控因子3114.2 细胞死亡细胞死亡概念：细胞死亡的一般定义是细胞生命现象不可逆的停止。细胞死亡有两种形式：一种为坏死性死亡，另一种为程序性死亡。3214.2.1 程序性细胞死亡及其特性程序性细胞死亡(programmed cell death, PCD), 又称细胞凋亡(apoptosis)是指为维持内环境稳定，由基因控制的细胞自主的有序性的死亡，它涉及一系列基因的激活、表达以及调控等的作用，因而是具有生理性和选择性的

24、。 Apoptosis：希腊语，是指树叶或花的自然凋落；33程序性细胞死亡34程序性死亡细胞的形态结构变化细胞变圆，染色质聚集、分块，胞质皱缩35程序性死亡细胞的DNA降解PCD生化特征：染色质DNA的有控裂解：核DNA在核小体连接处断裂成核小体片段，200bp的倍数DNA Ladder36细胞坏死与程序性细胞死亡凋亡小体 (apoptotic body)37比较内容程序性细胞死亡细胞坏死质膜不破裂发生破裂细胞核固缩，DNA片段化弥漫性降解细胞质由质膜包围溢出形成凋亡小体细胞破裂成碎片溶酶体的酶增多溶酶体解体蛋白质合成有无基因活动由基因调控无基因调控自吞噬常见缺少线粒体自身吞噬肿胀诱发因素生理

25、性信号强烈刺激信号对个体影响生长、发育、引起炎症生存所必需细胞坏死与程序性细胞死亡比较3814.2.2 程序性细胞死亡的机理2002年的诺贝尔生理学和医学奖:英国的Brenner、Sulston和美国的Horvitz，用C. elegans 研究了调控器官发育程序性细胞死亡的关键基因及其功能，并进一步在高等哺乳动物中发现了相关功能基因。Caenorhabditis elegans (C. elegans) 雌雄同体 39程序性细胞死亡的过程死亡激活期 (activation phase)：接收death signal死亡执行期 (execution phase)：执行一套死亡程序40 apop

26、tosis related genes in C elegans :决定死亡的两个基因，即ces-1(ces表示CE细胞存活的调控基因)和ces-2基因执行死亡的4个基因：ced-3、ced-4、ced-9和egl-1基因：“死亡机器”(death machinery)7个与死亡细胞被吞噬细胞所吞噬的基因，即ced-1、ced-2、ced-5、ced-6、ced-7、ced-10和ced-11。ced-9可抵消ced-3和ced-4 的作用，防止细胞被杀死，因此是存活因子; 死亡细胞在吞噬体中被降解的基因细胞凋亡的机理：基因调控作用的结果41Apoptotic genes in C. eleg

27、ans42 Caspase自杀性蛋白水解酶是天冬氨酸特异性半胱氨酸蛋白酶(cysteine-containing aspartate specific protease)，简称caspase;caspase-3、6、7和8 在FAS/TNF介导的程序性细胞死亡途径中起作用；caspase-9和3 一起参与线粒体中Apaf-I、细胞色素c介导的程序性细胞死亡;在人类，已经鉴定了10种不同的caspase。Apoptotic genes in mammalian cells43Apoptotic protease cascade in mammalian cells自杀性蛋白酶家族自我切割蛋白降解

28、级联44执行者caspase在程序性细胞死亡中的作用45能够被caspase切割的靶蛋白蛋白激酶核纤层蛋白细胞结构蛋白与DNA修复相关的酶类caspase激活的DNase抑制蛋白2022/9/2946Death receptors: CD95 (or Fas) TNFR1 (TNF receptor-1) DR4 and DR5. 细胞外信号（The extrinsic death pathway） 对程序性细胞死亡的激发47肿瘤坏死因子 （tumor necrosis factor, TNF）48细胞内信号（The intrinsic death pathway ）对程序性细胞死亡的激发内源

29、信号DNA损伤细胞质中Ca2+ 浓度过高极度氧胁迫（产生大量的自由基）正控制信号 促进细胞死亡,如细胞色素C，凋亡蛋白酶激活因子（apoptotic protease-activating factor，Apaf）负控制信号抑制细胞死亡，如哺乳动物中的BCL-2和BCL-x蛋白。49细胞内源信号激发细胞程序性死亡50 Apoptosis Regulators and effectors2022/9/2951Apoptotic proteins p53Bax线粒体外膜通透性2022/9/2952BidApoptotic proteins Bcl-2 FamilyBid2022/9/2953Apo

30、ptotic proteins Caspase family2022/9/2954Apoptotic proteins IAP family （inhibitor of apoptosis protein)livin2022/9/2955Apoptosis and senescence both are a failsafe (错误消除) mechanisms in cellCells respond to a number of potentially oncogenic stimuli by adopting a senescent or apoptosis, suggesting tha

31、t both are fail-safe mechanisms that protects cells from tumorigenic transformation. potentially oncogenic stimuliNormal SenescenceNormal ApoptosisNormal Cancer2022/9/2956Through accumulated genetic mutations, cell can be transformed, leading to tumors. Nature stops this tumorigenesis process throug

32、h apoptosis or senescence. However, as organisms age, the accumulation of senescent cells can create a pro-tumorigenic tissue environment. 2022/9/2957At the cellular level, activated p53 induced checkpoints in the cell-division cycle, permanent cell-division arrest (senescence) and cell death. At th

33、e whole-organism level, p53 activation results in a lower cancer incidence. But Tyner et al.3 show that p53 can also promote ageing. p53- common in apoptosis and senescence 2022/9/2958Apoptosis：a two-edged swordIn the reproductive years：providing critical tumor surveillancein a post reproductive per

34、iod：contributing to agingApoptosis and lifespan:Apoptosis vs Senescence lifespanApoptosis Senecence ( ageing )5914.2.3 程序性细胞死亡的意义动物机体靠对细胞增殖和细胞周期的正负控制以及对程序性细胞死亡的正负控制来维持细胞总数的平衡和机体的生命活力。程序性细胞死亡在形态建成中起重要作用。60动物细胞数量控制的途径61程序性细胞死亡在小鼠脚趾形成中的作用62蝌蚪向蛙发育的变态反应中程序性细胞死亡的作用63程序性细胞死亡对发育中神经细胞数量的调节竞争上岗64癌细胞所谓癌细胞实际上是一

35、种突变的体细胞，这种突变体脱离了细胞社会关于增殖和存活的控制，因此可以无限制的增殖产生肿瘤(Tumor)。分良性及恶性肿瘤。6514.1 癌生物学恶性肿瘤“癌”的类群癌(carcinoma)：上皮和内皮，内外胚层瘤(sarcoma)：结缔组织、肌肉，中胚层淋巴瘤(lymphoma)和白血病(leukemia)：是由淋巴和血液产生的癌，白血病主要是指癌细胞已经大量进入血液中。中胚层来源畸胎瘤（teratoma）早期胚胎细胞转化，有良恶之分癌细胞离体培养时接触抑制缺失癌细胞的生物学特性：无限增殖Colony formation/ transformation assay癌细胞的生物学特性：侵袭及转

36、移原位癌 vs 继发癌68癌细胞转移69PET全称为正电子发射计算机断层显像，是反映病变的基因、分子、代谢及功能状态的显像设备。它是利用正电子核素标记葡萄糖等人体代谢物作为显像剂，通过病灶对显像剂的摄取来反映其代谢变化，从而为临床提供疾病的生物代谢信息。Benign tumorMalignant tumor癌细胞恶性程度越高，分化程度越低快速增殖、不播散快速增殖、播散转移71 “癌”细胞形态外形：变圆细胞骨架结构紊乱核异常细胞质膜结构改变：细胞间连接、细胞表面受体癌细胞的染色体异常，如多倍体Pancreas cancer73“癌”细胞生理功能异常无限分裂、无接触抑制细胞黏着性、贴壁性减弱：Ce

37、ll-cell 、Cell-ECMeg：Fibronectin、cadherin、GAG易于凝集：凝集素74“癌”细胞生化异常细胞质膜成份改变：糖脂糖蛋白减少高尔基体成分变化：糖基转移酶缺乏纤连蛋白分泌减少新的膜抗原生成：MHA丢失生长因子需求降低分泌多种蛋白水解酶：MMPs75癌细胞的自分泌生长刺激7614.2 癌的起因： 物理和化学致癌物化学致癌物辐射对癌的诱发病毒77化学致癌物7814. 3 肿瘤发生遗传学癌发生的进程Normal cell Benign tumor Malignant tumor肿瘤抑制基因、癌基因与原癌基因79结肠癌的多步发展过程80癌基因与原癌基因癌基因(oncog

38、ene)：癌基因是细胞加速器，它们编码的蛋白使细胞生长不受控制，并促进细胞癌变细胞癌基因 (c-onc)：由细胞原癌基因突变而来；病毒癌基因 (v-onc)：大约已经鉴定了100多种不同的癌基因，它们中的大多数属于RNA肿瘤病毒基因 组中的基因。1976年发现，正常鸡细胞核DNA中，具有与V-onc的同源序列，称正常细胞中的同源序列为原癌基因（protooncogene）,或C-oncogene.v-onc和c-onc的关系：c-onc来自v-onc. 理由如下:1、v-onc对病毒复制和生存都是不必要的；而c-onc对细胞重要功能和活动不可缺少；提示c-onc是进化中保存下来的细胞重要结构元

39、件。2、发现的30种c-onc是依靠病毒的v-onc探针找到的；并不是所有c-onc都有对应的同源v-onc。3、v-src缺失3/4不能致癌的RSV,注入鸡体内，发现缺失的v-src与c-src发生重组，v-src回复并致癌。82原癌基因（proto-oncogene）:原本是细胞的正常基因，它们编码的蛋白质在正常细胞中通常参与细胞的生长与增殖的调节。但突变后成为促癌的癌基因(cancer-promoting oncogene)，导致细胞癌变。原癌基因突变成癌基因，称为原癌基因的激活。83原癌基因激活成癌基因84RNA肿瘤病毒癌基因起源的假说模型Proto-oncogenes Proto-oncogens: Gain-of-function mutationsRas oncogene: Retain bind GTPBcl-2 oncogene: Prevent apoptosis87癌基因转化肿瘤抑制基因(tumor suppressor gene)，抑癌基因（tumor suppressor gene）两个拷贝，只有当两个拷贝都丢失了或两个拷贝都失活了才会使细胞失去增殖的控制， 如：RB, P53Harris(1968):癌细胞系与同组织正常细胞融合杂交细胞无恶性表型，也不致癌； 随着染色体丢失则可能恢复致癌（Rb1