細(xì)胞生物學(xué)：chap15細(xì)胞衰老與死亡

1、Cell Aging and Cell DeathSection 1Cell aging/senescence Overview lIntroductionlThe lifespan of the celllChanges during senescencelTheories of aginglRelationship among age-related diseasesPart 1 Conception of Cell Aging1. Cell Aging and Organism agingCellular Aging Cellular Aging is defined as the no

2、rmal process accompanied by the progressive alteration in the bodys / cells homeostatic adaptive responses.Progressive time related loss of structural and functional capacity of cells leading to death.lSenescence is not the inevitable fate of all organisms. 機(jī)體衰老機(jī)體衰老是指絕大多數(shù)生物性成熟以后，機(jī)是指絕大多數(shù)生物性成熟以后，機(jī)體形態(tài)結(jié)

3、構(gòu)和生理功能逐漸退化或老化的過(guò)體形態(tài)結(jié)構(gòu)和生理功能逐漸退化或老化的過(guò)程，是一個(gè)受發(fā)育程序、環(huán)境因子等多因素程，是一個(gè)受發(fā)育程序、環(huán)境因子等多因素控制的不可逆的生物學(xué)現(xiàn)象，機(jī)體的衰老與控制的不可逆的生物學(xué)現(xiàn)象，機(jī)體的衰老與動(dòng)物的壽命密切相關(guān)。動(dòng)物的壽命密切相關(guān)。lSenescence occurs both on the level of individual cells (cellular senescence) as well as on the level of the whole organism. 4organismal aging（機(jī)體衰老）機(jī)體衰老） u個(gè)別細(xì)胞或局部許多細(xì)胞的衰老

4、并不影響機(jī)個(gè)別細(xì)胞或局部許多細(xì)胞的衰老并不影響機(jī)體的壽命體的壽命u機(jī)體衰老并不代表所有細(xì)胞的衰老機(jī)體衰老并不代表所有細(xì)胞的衰老u個(gè)體的衰老是建立在總體細(xì)胞衰老的基礎(chǔ)上個(gè)體的衰老是建立在總體細(xì)胞衰老的基礎(chǔ)上u細(xì)胞總體的衰老反應(yīng)了機(jī)體的衰老細(xì)胞總體的衰老反應(yīng)了機(jī)體的衰老5細(xì)胞衰老與機(jī)體衰老的關(guān)系細(xì)胞衰老與機(jī)體衰老的關(guān)系lDifferent cells show different lifespan.lThe differentiated level and dividing potency related to lifespan of the cell.（1）長(zhǎng)壽命細(xì)胞）長(zhǎng)壽命細(xì)胞 （細(xì)胞壽命接近

5、整體壽命）（細(xì)胞壽命接近整體壽命） （2）緩慢更新的細(xì)胞）緩慢更新的細(xì)胞（3）快速更新且壽命較短的細(xì)胞）快速更新且壽命較短的細(xì)胞Proliferative lifspan is related to the donors age2. The different lifespan of the cellCellular senescence / aginglCells from old donors divide less often than cells from young donors.lCells from short-lived species are more sensitive t

6、o senescence-inducers, particularly oxidative stress, than cells from long-lived species.lCells from donors with premature aging syndromes senesce more readily than cells from normal donors.lSenescent cells (expressing a senescence marker) accumulate with age and at sites of age-related pathology.3.

7、 The lifespan of the cultured cell in vitro 體外培養(yǎng)的細(xì)胞壽命取決于體外培養(yǎng)的細(xì)胞壽命取決于 1）體外可傳代的次數(shù)）體外可傳代的次數(shù) 2）來(lái)源個(gè)體的年齡）來(lái)源個(gè)體的年齡 3）物種的壽命）物種的壽命Senescense inducers l Genetic Clock genesl Diet malnutrition, obesity etc.l Social conditions Diseases Atherosclerosis, diabetes etc.l Werners syndromel Hutchinson-Gilford progeria

8、 syndromeThe Hayflick Limit:Hayflick and Moorhead, 1961 They noticed that fibroblast cultures stopped dividing after an average of 50 cumulative population doublings.This phenomenon of growth arrest after a period of apparently normal cell proliferation is known as the Hayflick limit (or Hayflick ph

9、enomenon) .Hayflick L., Moorhead P.S. The serial cultivation of human diploid cell strains, Exp. Cell Res., 1961, v. 253: 585-621.The Hayflick limitProliferative capacityNumber of cell divisionsFiniteReplicativeLife SpanMortalInfiniteReplicativeLife SpanImmortalEXCEPTIONS Germ line； ； Embryonic stem

10、 cells； ；Tumor cellsThe lifespan of the cell+What causes cellular senescence?決定細(xì)胞衰老的因素在細(xì)胞內(nèi)部，而不是外部的環(huán)境決定細(xì)胞衰老的因素在細(xì)胞內(nèi)部，而不是外部的環(huán)境; 老年男性體細(xì)胞老年男性體細(xì)胞 混合培養(yǎng)混合培養(yǎng) 年輕女性體細(xì)胞年輕女性體細(xì)胞是細(xì)胞核而不是細(xì)胞質(zhì)決定了細(xì)胞衰老是細(xì)胞核而不是細(xì)胞質(zhì)決定了細(xì)胞衰老Cytochalasin處理細(xì)胞處理細(xì)胞 得到胞質(zhì)體得到胞質(zhì)體離心離心年輕女性體細(xì)胞年輕女性體細(xì)胞老年輕男性體細(xì)胞老年輕男性體細(xì)胞Part 2 The appearances of aging cellsl

11、Cell large, flat, and vacuole-rich cytoplasmlChanges of nucleusNuclear membrane invagination MultinucleatedlChanges of ER and mitochondrialChanges of cell membranelResidual and pigments1. Morphological changes of aging cellsDNA： break down, degrades, crosslink, DNA methylation decreases RNA： break d

12、own, especially rRNA, mRNA, tRNA Protein synthesis decreases and its degradation increases Enzyme activity decreases Lipid: peroxidation, fluidity decreases and cohesion increase 2. Biochemical changes of aging cells15 衰老癥狀與原因分析衰老癥狀與原因分析皮膚干燥、發(fā)皺皮膚干燥、發(fā)皺頭發(fā)變白頭發(fā)變白老人斑老人斑飲食減少飲食減少細(xì)胞水分減少，體積減小細(xì)胞水分減少，體積減小細(xì)胞內(nèi)的酶

13、活性降低細(xì)胞內(nèi)的酶活性降低細(xì)胞內(nèi)色素的累積細(xì)胞內(nèi)色素的累積細(xì)胞膜通透性功能改變細(xì)胞膜通透性功能改變Part 3 The mechanism of cell aginglMost scientists now agree that aging is, at least in part, the result of accumulating damage to the moleculessuch as proteins, lipids, and nucleic acids.l Aging is a result of internal or external assaults that damag

14、e cells or organs so they can no longer function properly. Many theories are a combination of programmed and error theories.1. The theories of cell aging 子女的壽命與雙親的壽命有關(guān)；子女的壽命與雙親的壽命有關(guān)； 各種動(dòng)物都有相當(dāng)恒定的平均壽命和最高壽命；各種動(dòng)物都有相當(dāng)恒定的平均壽命和最高壽命； 細(xì)胞衰老時(shí)，衰老相關(guān)基因表達(dá)活性增高；細(xì)胞衰老時(shí)，衰老相關(guān)基因表達(dá)活性增高； MORF4，p16 抗衰老基因（長(zhǎng)壽基因抗衰老基因（長(zhǎng)壽基因longe

15、vity gene）突變引致）突變引致細(xì)胞衰老；細(xì)胞衰老；早衰癥患者體內(nèi)解旋酶（早衰癥患者體內(nèi)解旋酶（WRN基因）發(fā)生突變。基因）發(fā)生突變。酵母酵母sgs1基因（與基因（與WRN基因同源）突變體壽命明顯短于野基因同源）突變體壽命明顯短于野生型。生型。 Caenrhabditis elegans的平均壽命僅的平均壽命僅3.5天，該蟲(chóng)天，該蟲(chóng)age-1 單基因突變，可提高平均壽命單基因突變，可提高平均壽命65%，提高最大壽命，提高最大壽命110%。 （1）Genetic Determined Theory lAging in Fast-Forward: Werner Syndrome lMary

16、was diagnosed with Werner syndrome at age 26. In her early 20s, she noticed her hair graying and falling out, and her skin became unusually wrinkled for someone her age. Soon after the diagnosis, she developed diabetes. Hutchinson-Gilford progeria syndrome（ Lamin A ：LMNA）At age 15, this Japanese-Ame

17、rican woman looked healthy, but by age 48, she had clearly developed symptoms of Werner syndrome. （WRN）Population DoublingsPremature senescence of Mof-/- MEFs小鼠胚胎成纖維細(xì)胞小鼠胚胎成纖維細(xì)胞(MEF)l A free radical is a molecule with an unpaired, highly reactive electron. Exogenous free radical: : be produced as a r

18、esult of environmental pollution. Endogenous free radical: be produced during metabolism l Reactive oxygen species can damage many kinds of biological molecules. can mutate genesdamage the lipids break the proteins (2) Free Radical TheoryAntioxidants，such as vitamins E and C，they can disarm dangerou

19、s reactive oxygen compounds. Telomere:l It is made up of thousands of building blocks with a sequence TTAGGG that repeats over and over. l Protects the chromosomal terminal from fusion and degradation.(3) Telomeres: Cellular Timekeepers l Telomere lengths are maintained by the enzyme telomerase, whi

20、ch is expressed by cells that comprise the germline as well as many cancer cells. TelomereHow chromosomes are protected by telomeres and the enzyme telomeraseThe Nobel Prize in Physiology or Medicine 2009 was awarded jointly to them for the discovery of how chromosomes are protected by telomeres and

21、 the enzyme telomerase.Elizabeth H. Blackburn Carol W. Greider Jack W. SzostakBlackburn and Szostak discovered that a unique DNA sequence in the telomeres protects the chromosomes from degradation. Greider and Blackburn identified telomerase.(4) The other theoriesl細(xì)胞代謝廢物累積可細(xì)胞代謝廢物累積可引起細(xì)胞衰老引起細(xì)胞衰老l基因轉(zhuǎn)錄

22、或翻譯差錯(cuò)基因轉(zhuǎn)錄或翻譯差錯(cuò)導(dǎo)致細(xì)胞衰老導(dǎo)致細(xì)胞衰老l其他其他The aging process is driven by a lifelong accumulation of molecular damage, resulting in gradual increase in the fraction of cells carrying defects. After sufficient time has passed, the increasing levels of these defects interfere with both the performance and functio

23、nal reserves of tissues and organs, resulting in age-related frailty, disability, and disease. Stress, adverse environment, and poor nutrition can increase the rate at which molecular damage arises. Intrinsic maintenance mechanisms, such as DNA repair and antioxidants, can slow the rate of accumulat

24、ion.2. The pathway of cell agingl復(fù)制性衰老復(fù)制性衰老 pRbl氧化應(yīng)激誘導(dǎo)的非端粒性衰老氧化應(yīng)激誘導(dǎo)的非端粒性衰老(premature senescence) ERKp38MAPK p16 pRblThe cells are no longer capable of dividing but these cells will remain metabolically active. This phenomenon is also known as replicative senescence“.Part 4 Relationship among cell ag

25、ing and diseasesAlzheimers disease (AD), Parkinsons disease (PD) and Huntingtons disease (HD)lloss-of-function pathologies, are dengenerative in naturelgain-of-function pathologies, are generally hyperplastic in natureCellular SenescenceAn important tumor suppressor mechanismlInduced by potentially

26、oncogenic events -oncogenes- RaslMost tumor cells are immortallMany oncogenes act by allowing cells to bypass the senescence responselSenescence is controlled by the two most important -tumor suppressor genes - p53 and pRBSection 2Cell deathOverview1. Differences: necrosis vs. apoptosis2. Morphologi

27、cal changes3. Role of apoptosis in diseases 4. Biochemical mechanisms 5. Detection of apoptosis細(xì)胞死亡（細(xì)胞死亡（cell death）指細(xì)胞生命現(xiàn)象的終結(jié)）指細(xì)胞生命現(xiàn)象的終結(jié). Types of cell death:細(xì)胞壞死細(xì)胞壞死(necrosis)是指在外來(lái)致病因子的作用下，是指在外來(lái)致病因子的作用下，細(xì)胞生命活動(dòng)被強(qiáng)行終止所致的病理性、被動(dòng)性的細(xì)胞生命活動(dòng)被強(qiáng)行終止所致的病理性、被動(dòng)性的死亡過(guò)程。死亡過(guò)程。 細(xì)胞壞死時(shí)，細(xì)胞膜與胞漿中的細(xì)胞器的質(zhì)膜發(fā)生破細(xì)胞壞死時(shí)，細(xì)胞膜與胞漿中的細(xì)胞器

28、的質(zhì)膜發(fā)生破裂，細(xì)胞漿外溢，細(xì)胞解體并引起周?chē)M織發(fā)生炎癥裂，細(xì)胞漿外溢，細(xì)胞解體并引起周?chē)M織發(fā)生炎癥反應(yīng)。反應(yīng)。 Necrosis is accidental type of death.細(xì)胞凋亡細(xì)胞凋亡(apoptosis)是指在特定信號(hào)誘導(dǎo)下，細(xì)是指在特定信號(hào)誘導(dǎo)下，細(xì)胞內(nèi)的死亡級(jí)聯(lián)反應(yīng)被觸發(fā)所致的生理或病理性胞內(nèi)的死亡級(jí)聯(lián)反應(yīng)被觸發(fā)所致的生理或病理性、主動(dòng)性的死亡過(guò)程。、主動(dòng)性的死亡過(guò)程。 細(xì)胞凋亡發(fā)生時(shí)，細(xì)胞包被成一些囊狀小體，即細(xì)胞凋亡發(fā)生時(shí)，細(xì)胞包被成一些囊狀小體，即凋凋亡小體亡小體（apoptotic body),后者被周?chē)淌杉?xì)胞吞后者被周?chē)淌杉?xì)胞吞噬，不引起炎癥。噬，不

29、引起炎癥。Part 1 The reasons,characteristics and forms of cell death細(xì)胞壞死細(xì)胞壞死(necrosis)；細(xì)胞凋亡細(xì)胞凋亡(Apoptosis)Differences: necrosis vs. apoptosis壞死細(xì)胞壞死細(xì)胞凋亡細(xì)胞凋亡細(xì)胞NecrosisNecrosislNecrosis refers to a spectrum of morphologic changes that follow cell death in living tissue. It is a passive process. Necrosis can

30、 damage surrounding cells and frequently cause inflammation.l Causes: - mechanical Damage - exposure to toxic chemicals - infectionlResults: - membrane permeabilizes - cell swells - DNA degraded - leaking out of cell contents - inflammation of surrounding tissuesPart 2 The characteristics of cell ap

31、optosislcell apoptosis-programmed cell death(PCD)-carefully planned (suicide)lThe expression apoptosis has been coined by Kerr et al.(1972) to describe a specific morphological aspect of cell death.lThe term apoptosis should be applied exclusively to cell death events that occur while manifesting se

32、veral among these morphological features. It is worth noting that it is not correct to assume that programmed cell death (PCD) and apoptosis are synonyms because cell death, as it occurs during physiological development, can manifest non-apoptotic features.Definition of apoptosisApoptosisl Causes:Wi

33、thdrawal of surviving signals (e.g. growth factors)Receipt of death signals (death activators, signals fromDNA damage, increased oxidant levels etc.)l Results: -shrink -bubble-like blebs on cell surface -Chromatin condensation and DNA degradation -mitochondria break down -break into small, membrane-

34、wrapped, fragments. -Externalization of phospholipids (e.g. phosphatidylserine) - With no inflammationProgrammed Cell Death lIn a multicellular ,if cells are no longer needed, they commit suicide by activating an intracellular death program. This process is therefore called programmed cell death.l程序

35、性細(xì)胞死亡程序性細(xì)胞死亡( PCD): 為維持內(nèi)環(huán)境穩(wěn)定，為維持內(nèi)環(huán)境穩(wěn)定，在在一定時(shí)間內(nèi)，細(xì)胞按一定的程序發(fā)生死亡，這一定時(shí)間內(nèi)，細(xì)胞按一定的程序發(fā)生死亡，這種細(xì)胞死亡具有嚴(yán)格的基因時(shí)控性和選擇性。種細(xì)胞死亡具有嚴(yán)格的基因時(shí)控性和選擇性。Functions of PCD during DevelopmentApoptosis is as needed for proper development1. The morphological changes of Apoptosis（1）細(xì)胞核：）細(xì)胞核：核核DNA聚集，濃縮成染色質(zhì)塊，細(xì)胞核呈帽狀聚集，濃縮成染色質(zhì)塊，細(xì)胞核呈帽狀或新月形，染色質(zhì)

36、進(jìn)一步聚集使核膜斷裂，并形成核碎片或或新月形，染色質(zhì)進(jìn)一步聚集使核膜斷裂，并形成核碎片或核殘片。核殘片。（2 2）細(xì)胞質(zhì)：）細(xì)胞質(zhì)：細(xì)胞質(zhì)密度增加，細(xì)胞質(zhì)密度增加，胞漿濃縮。胞漿濃縮。線粒體先增大，線粒體先增大，而后空泡化；內(nèi)質(zhì)網(wǎng)囊腔膨脹，并逐漸與質(zhì)膜融合；細(xì)胞骨而后空泡化；內(nèi)質(zhì)網(wǎng)囊腔膨脹，并逐漸與質(zhì)膜融合；細(xì)胞骨架致密并紊亂。架致密并紊亂。（3 3）細(xì)胞膜：）細(xì)胞膜：微絨毛、細(xì)胞突起、細(xì)胞間連接復(fù)合體普遍消微絨毛、細(xì)胞突起、細(xì)胞間連接復(fù)合體普遍消失。此時(shí)，細(xì)胞膜結(jié)構(gòu)仍保持完整。失。此時(shí)，細(xì)胞膜結(jié)構(gòu)仍保持完整。（4 4）凋亡小體的形成：）凋亡小體的形成：細(xì)胞核的碎片形成后會(huì)不斷轉(zhuǎn)化為大細(xì)胞核的

37、碎片形成后會(huì)不斷轉(zhuǎn)化為大小不等，內(nèi)含胞質(zhì)，細(xì)胞器以及核碎片的膜包小體，即小不等，內(nèi)含胞質(zhì)，細(xì)胞器以及核碎片的膜包小體，即凋亡凋亡小體。小體。ApoptosisProgression of apoptosisMulticellular organisms eliminate redundant, damaged or infected cells by a process of cell suicide.TEM of a cell in late apoptosis shows radical changes in cell shape, with membrane blebbing and t

38、he formation of many membrane-bound cytoplasmic regions. These apoptotic bodies may separate from one another but remain enclosed by plasma membrane so that no contents are released into the extracellular space. The membrane changes are recognized by neighboring cells, and macrophages and apoptotic

39、bodies are very rapidly phagocytosed. X10,000.Examples of isolated nonapoptotic and apoptotic adult rat ventricular myocytes in culture. Note the condensation of nuclear chromatin (pyknosis), nuclear fragmentation (karyohexis), and membrane blebbing in the apoptotic cells. Image prepared using Tunne

40、l staining. Reprinted with permission from Pinsky DJ, Aji W, Szabolcs M, et al. Nitric oxide triggers programmed cell death (apoptosis) of adult rat ventricular myocytes in culture. Am J Physiol. 1999;277:H1189-H1199.(1)DNA 片段化（片段化（fragmentation)l DNA is broken down into 50 to 300 kilobase pieces梯狀條

41、梯狀條帶帶（DNA ladders）l Subsequently, there is internucleosomal cleavage of DNA into oligonucleosomes, in multiples of 180 to 200 base pairs, by Ca2+ and Mg2+ dependent endonucleases(核酸內(nèi)核酸內(nèi)切酶切酶, ,ZnZn2+2+是其抑制劑）是其抑制劑）2. The biochemical changes of Apoptosis(2) (2) 細(xì)胞凋亡中的蛋白酶細(xì)胞凋亡中的蛋白酶 控制凋亡的蛋白酶有多種，如控制凋亡的蛋白酶有

42、多種，如胱天蛋白酶（胱天蛋白酶（cystein aspartic acid specific protease, Caspase) ，鈣蛋白酶。，鈣蛋白酶。lActive caspases cleave many vital cellular proteins, such as lamins, and thus breakup nuclear scaffold and cytoskeleton.lCaspases also activate DNAses, which degrade nuclear DNA.(3)(3)胞漿胞漿CaCa2+ 2+ 、pHpH的變化的變化Ca2+庫(kù)開(kāi)放庫(kù)開(kāi)放，胞

43、內(nèi)，胞內(nèi)Ca2+濃度升高，可作為凋亡啟動(dòng)的信號(hào)。濃度升高，可作為凋亡啟動(dòng)的信號(hào)。Ca2+的釋放會(huì)破壞細(xì)胞的穩(wěn)定結(jié)構(gòu)，從而也會(huì)觸發(fā)細(xì)胞凋亡。的釋放會(huì)破壞細(xì)胞的穩(wěn)定結(jié)構(gòu)，從而也會(huì)觸發(fā)細(xì)胞凋亡。細(xì)胞凋亡時(shí)，胞漿內(nèi)的細(xì)胞凋亡時(shí)，胞漿內(nèi)的pH先急性升高，后又緩慢降低。先急性升高，后又緩慢降低。(4) (4) 線粒體在細(xì)胞凋亡中的生化變化線粒體在細(xì)胞凋亡中的生化變化1）線粒體呼吸鏈?zhǔn)軗p，）線粒體呼吸鏈?zhǔn)軗p，能量代謝受到破壞，導(dǎo)致細(xì)胞死亡。能量代謝受到破壞，導(dǎo)致細(xì)胞死亡。2）線粒體釋放細(xì)胞色素）線粒體釋放細(xì)胞色素C（cytochrome C, cytC) 而而cytC是是凋亡所必需的胱天蛋白酶凋亡所必需的胱

44、天蛋白酶(caspase)家族激活物。家族激活物。3）線粒體是細(xì)胞產(chǎn)生活性氧類(lèi)物質(zhì)（）線粒體是細(xì)胞產(chǎn)生活性氧類(lèi)物質(zhì)（reactive oxygen species, ROS)的主要來(lái)源，的主要來(lái)源，ROS是細(xì)胞凋亡的信使分子和效是細(xì)胞凋亡的信使分子和效應(yīng)分子，凋亡時(shí)，線粒體生成應(yīng)分子，凋亡時(shí)，線粒體生成ROS增多。增多。4）線粒體滲透轉(zhuǎn)變孔（）線粒體滲透轉(zhuǎn)變孔（permeability transition pore, PT pore）通透性增高，這是凋亡早期的決定性變化。通透性增高，這是凋亡早期的決定性變化。PT孔是線孔是線粒體內(nèi)膜和外膜在接觸部位協(xié)同組成的一條通道，粒體內(nèi)膜和外膜在接觸部位

45、協(xié)同組成的一條通道，PT孔的開(kāi)孔的開(kāi)放可導(dǎo)致線粒體呼吸鏈解偶聯(lián)，并且線粒體內(nèi)的放可導(dǎo)致線粒體呼吸鏈解偶聯(lián)，并且線粒體內(nèi)的cyt C可通過(guò)可通過(guò)開(kāi)放開(kāi)放PT孔釋放到胞質(zhì)，進(jìn)而觸發(fā)孔釋放到胞質(zhì)，進(jìn)而觸發(fā)caspase 級(jí)聯(lián)反應(yīng)。級(jí)聯(lián)反應(yīng)。 細(xì)胞凋亡細(xì)胞凋亡時(shí)時(shí)的線粒體釋放細(xì)胞色素的線粒體釋放細(xì)胞色素C3. Anoikis (失巢凋亡失巢凋亡)Part 3 The influencing factors of cell apoptosis生理性誘導(dǎo)因子生理性誘導(dǎo)因子: TNF , FasL, TGF-，神經(jīng)遞質(zhì)、，神經(jīng)遞質(zhì)、 Ca2+, 糖皮質(zhì)激素等。糖皮質(zhì)激素等。 損傷相關(guān)因子損傷相關(guān)因子: 熱休

46、克，病毒感染，原癌基因、熱休克，病毒感染，原癌基因、 抑癌基因、抑癌基因、 細(xì)胞毒性細(xì)胞毒性T細(xì)胞，氧化劑、自由基細(xì)胞，氧化劑、自由基. 缺血、缺氧等。缺血、缺氧等。 疾病治療相關(guān)因子疾病治療相關(guān)因子: 化療，化療， 放療，等放療，等 其它某些細(xì)胞毒性物質(zhì)其它某些細(xì)胞毒性物質(zhì): 如乙醇、等。如乙醇、等。1.細(xì)胞凋亡的誘發(fā)因素細(xì)胞凋亡的誘發(fā)因素山東大學(xué)醫(yī)學(xué)院細(xì)胞生物學(xué)研究所54生理性抑制因子生理性抑制因子: 如如bcl-2 原癌基因，突變型原癌基因，突變型p53,各種生長(zhǎng)因子，細(xì)胞外基質(zhì)。各種生長(zhǎng)因子，細(xì)胞外基質(zhì)。 病毒基因病毒基因: 如腺病毒如腺病毒E1B 等等 其它其它: 線蟲(chóng)的線蟲(chóng)的ced

47、-9 基因，基因，caspase 抑制劑，促抑制劑，促癌劑（如癌劑（如PMA）。）。2. 細(xì)胞凋亡的抑制因素細(xì)胞凋亡的抑制因素Inducers of ApoptosisTNF familyFas LigandTNFTGFNeurotransmitters GlutamateDopamineN-methyl-D-aspartateGrowth Factor withdrawalLoss of matrix attachmentCalcium Glucocorticoids Heat ShockViral InfectionBacterial ToxinsOncogenesmyc,rel, E1A

48、tumor suppressors p53Cytolytic T cellsOxidantsFree RadicalsNutrient Deprivationanti-metabolitesChemotherapeutic drugscisplatin, doxorubicinbleomycin, cytosinearabinoside, nitrogenmustard, methotrexate,vincristineGamma-radiationUV-radiationPhysiological Activators Damage-Related Activators Therapy-as

49、sociated AgentsPart 4 The molecular mechanism of cell apoptosisStage 1 - The Death SignalStage 2 - Integration and TransductionStage 3 - ExecutionStage 4 Cell RemovalThe Nobel Prize in Physiology or Medicine 2002for their discoveries concerning genetic regulation of organ development and programmed

50、cell deathSydney Brenner H. Robert Horvitz John E. Sulston The Molecular Sciences Institute Berkeley, CA, USA Massachusetts Institute of Technology (MIT) Cambridge, MA, USA The Wellcome Trust Sanger Institute Cambridge, United Kingdom During development, 1090 cells are generated, but precisely 131 o

51、f these cells are eliminated by programmed cell death. This results in an adult nematode (the hermaphrodite), composed of 959 somatic cells. 1. gene regulation of cell apoptosis Leaders in the field of programmed cell death (apoptosis) Stanley J. Korsmeyer ( 1950 2005) Korsmeyer did pioneering work

52、on the regulation of apoptosis. He identified the key genetic mechanisms that govern cell death and survival and defined the role of cell death in the pathogenesis of human diseases.王曉東王曉東 (Xiaodong Wang) 北京生命科學(xué)研究所所長(zhǎng)、資深研究員 美國(guó)國(guó)家科學(xué)院院士袁鈞英袁鈞英 (Junying Yuan)Fellow of AAASProfessor of Cell BiologyHarvard

53、Medical School(1) Ced基因家族基因家族 Ced 基因是線蟲(chóng)的細(xì)胞凋亡關(guān)鍵調(diào)控基因。基因是線蟲(chóng)的細(xì)胞凋亡關(guān)鍵調(diào)控基因。 l Ced3 、ced4基因促進(jìn)細(xì)胞的凋亡?；虼龠M(jìn)細(xì)胞的凋亡。l Ced9基因可抑制細(xì)胞凋亡的發(fā)生基因可抑制細(xì)胞凋亡的發(fā)生l與與Ced基因家族同源的哺乳動(dòng)物基因基因家族同源的哺乳動(dòng)物基因 Caspase-3與與Ced3同源同源 Apa-f1與與Ced4同源同源 Bcl-2基因家族基因家族與與Ced9有一定的同源性有一定的同源性Regulation of Ced Proteins for Cell Apoptosis當(dāng)細(xì)胞膜表面的死亡受體被死亡信號(hào)激活時(shí)，當(dāng)

54、細(xì)胞膜表面的死亡受體被死亡信號(hào)激活時(shí)，Ced9蛋白呈現(xiàn)非活性狀態(tài)，蛋白呈現(xiàn)非活性狀態(tài)，Ced-3 和和 Ced-4分子發(fā)揮活性，誘導(dǎo)細(xì)胞發(fā)生凋亡。分子發(fā)揮活性，誘導(dǎo)細(xì)胞發(fā)生凋亡。哺乳動(dòng)物哺乳動(dòng)物 參與細(xì)胞凋亡的參與細(xì)胞凋亡的Caspase基因基因 lCaspase活性位點(diǎn)是半胱氨酸，裂解靶蛋白位點(diǎn)是天冬氨酸殘基后的肽鍵，因此稱(chēng)為Cysteine Aspartic Acic Specific Protease，即Caspase。lCed4的哺乳類(lèi)同源物直到1997年，才被證明是Apaf-1（apoptosis protease activating factor）。lCed 9的哺乳類(lèi)對(duì)應(yīng)物被證

55、明是BCL-2。凋亡起始分子凋亡起始分子凋亡效應(yīng)分子凋亡效應(yīng)分子 參與凋亡執(zhí)行，參與凋亡執(zhí)行， 它們能降它們能降解多種底物，導(dǎo)致核纖層解多種底物，導(dǎo)致核纖層和細(xì)胞骨架的斷裂崩解。和細(xì)胞骨架的斷裂崩解。細(xì)胞因子前體細(xì)胞因子前體Evolutionary Conservation of theCore Apoptotic MachineryCommon intracellular machinery for apoptosisThe three main playersa. Family of enzymes - Caspasesb. Protein family - Bcl-2 proteinsc

56、. Regulating gene - p53 geneprodomainlarge subunitssmall subunitscleavagesitesProcaspase structureCaspase inhibitors IAP, inhibitors of apoptosis proteins 細(xì)胞內(nèi)存在的細(xì)胞內(nèi)存在的Caspase抑制蛋白，抑制蛋白，IAP inhibitors of apoptosis proteins家族，阻止細(xì)胞凋亡過(guò)程。家族，阻止細(xì)胞凋亡過(guò)程。（2）bcl-2 gene familyBcL-2基因家族成員基因家族成員自身或彼此之間有形成自身或彼此之間有形

57、成二聚體或多聚體的能力，二聚體或多聚體的能力，BcL-2家族的蛋白與蛋家族的蛋白與蛋白相互作用調(diào)節(jié)著細(xì)胞白相互作用調(diào)節(jié)著細(xì)胞的存活與凋亡。的存活與凋亡。Molecular Structure of Bcl-2 Protein FamilyBcl-2 proteins and apoptosislTo regulate mitochondrial permeability (bind to the outer mem. of mit.) lAnti-apoptotic Bcl: cell survival stimulus-induced lPro-apoptotic Bcl: death si

58、gnal-inducedl1993年Yuan從哺乳類(lèi)細(xì)胞中發(fā)現(xiàn)第1個(gè)Ced-3同源分子，叫白細(xì)胞介素-1轉(zhuǎn)換酶（Interleukin-1 Converting Enzyme，ICE），Pro-ICE可自身催化產(chǎn)生二個(gè)片段，聚合形成雜四聚體，為活化的ICE。 其作用是將其作用是將IL-1前體分解為有生理活性的白細(xì)胞介素前體分解為有生理活性的白細(xì)胞介素-1。 ICE過(guò)度表達(dá)可誘導(dǎo)哺乳動(dòng)物纖維母細(xì)胞發(fā)生凋亡。過(guò)度表達(dá)可誘導(dǎo)哺乳動(dòng)物纖維母細(xì)胞發(fā)生凋亡。l人類(lèi)共發(fā)現(xiàn)有14個(gè)Ced-3同源分子，分別定名為Caspase1-14。（3）ice基因基因（4）p53 gene and p53 protein

59、lTumour suppressor gene lActivated in response to DNA damagelPrevents cell completing cell cycle If damage is minor - allows repairIf major - induces apoptosislComplex mechanisms 野生型：促進(jìn)細(xì)胞凋亡野生型：促進(jìn)細(xì)胞凋亡 突變型突變型: 抑制細(xì)胞凋亡抑制細(xì)胞凋亡p53lFas 屬于腫瘤壞死因子受體（屬于腫瘤壞死因子受體（tumor necrosis factor receptor, TNFR)和神經(jīng)生長(zhǎng)因子受體和神經(jīng)生

60、長(zhǎng)因子受體(nerve growth factor receptor, NGFR） 超家族的細(xì)胞表面分子；超家族的細(xì)胞表面分子；lFas配體配體（Fas ligand, FasL)是是TNF家族的細(xì)胞表面分子。家族的細(xì)胞表面分子。l多種哺乳動(dòng)物細(xì)胞表達(dá)多種哺乳動(dòng)物細(xì)胞表達(dá)Fas；FasL只在活化的只在活化的T細(xì)胞表達(dá)細(xì)胞表達(dá)lFas需要通過(guò)和需要通過(guò)和FasL結(jié)合后發(fā)揮調(diào)控作用結(jié)合后發(fā)揮調(diào)控作用 。（5）可觸發(fā)細(xì)胞凋亡的可觸發(fā)細(xì)胞凋亡的Fas and FasLMolecular Structure of Fas配體FasL與死亡受體Fas結(jié)合后，誘導(dǎo)Fas胞質(zhì)區(qū)內(nèi)的死亡結(jié)構(gòu)域（DD）結(jié)合Fas