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1、第8章 恶性肿瘤的分子生物学(Molecular Biology of Malignant Tumors),马立克氏病病鸡,图1 卵巢肿瘤A：GAe组雏鸡接种MDV GA株后40天，卵巢形成明显肿瘤（如箭头所示）；B：GA0组卵巢正常（如箭头所示）；C：异常卵巢和正常卵巢的直观比较）,图2 睾丸肿瘤A：GA0组睾丸正常（如箭头所示）；B：GAe组雏鸡接种MDV GA株后40天，睾丸形成明显肿瘤（如箭头所示）；C：异常睾丸和正常睾丸的直观比较）,肿瘤(tumor)是机体在各种致瘤因素作用下，局部组织的细胞在基因水平上失去对其生长的正常调控，导致克隆性异常增生而形成的新生物,9.1 概述,肿瘤有良
2、性、恶性之分。仅快速增殖而不扩散的称为良性肿瘤；而不仅异常快速增殖，而且细胞可发生扩散转移的称为恶性肿瘤，特称为癌（cancer）或新生物（neoplasm）。癌的主要特征：不受机体整体的制约，“为所欲为”地快速增殖。除了侵占正常细胞所占的空间外，还发生扩散和转移，形成新的病灶。,Benign tumors arise with great frequency but pose little risk because they are localized and small,Malignant tumors generally invade surrounding tissue and spr
3、ead throughout the body,Alterations in cell-cell interactions and the formation of new blood vessels are associated with malignancy,恶性肿瘤的转移,对肿瘤发生的分子机制，最初人们认为是病毒瘤基因所致；70年代中期，毕晓普和瓦慕斯发现动物正常细胞中本来存在癌基因（原癌基因）；正常情况下，瘤基因对细胞的生长、分化、调节和维护正常功能起重要作用；近来发现细胞中有抑制肿瘤发生的抑瘤基因。,1989年诺贝尔生理学或医学奖,分子肿瘤学认为恶性肿瘤是一种涉及基因改变的疾病,Fo
4、r cancer:two classes of cellular genes are targets for mutations,PROTO-ONCOGENESTUMOUR SUPRESSOR GENES,The vast majority of these mutations are somatic,Balance of Tissue Cell Number,Oncogenes(癌基因),Normal genes(regulate cell growth),1st mutation(leads to accelerated cell division),1 mutation sufficie
5、nt for role in cancer development,ExampleOncogenes derived from growth factor receptors confer the ability to bypass the growth factor requirementleading to independent growth.,Tumour suppressor genes（肿瘤抑制基因）,Act as a brake for cell division,“GUARDIAN OF THE GENOME”,PROBLEM:Mutation in tumour suppre
6、ssor genes=brakes dont work,or there is an accumulations of mutations(DNA repair enzymes),The mutated gene product is not functional=NULL ALLELE,Inactivated tumour suppressor gene,肿瘤发生,Activated proto-oncogene,癌基因(oncogeny，onc)是指能导致细胞恶性转化的核酸片段。主要包括病毒癌基因、细胞癌基因 以及与细胞生长因子及其受体、蛋白激酶、转录因子及其信息加工、传递等有关的基因。,
7、9.2 几个基本概念,病毒癌基因（virus oncogenes,V-onc）：存在于病毒基因组内的癌基因，病毒感染动物时，可诱导动物体内肿瘤形成。DNA病毒RNA病毒（反转录病毒）细胞癌基因（cellular oncogenes,C-onc）：存在于动物细胞中的癌基因。正常情况下主要在胚胎期表达，在出生后的个体内不表达或限量表达，主要功能是促进细胞的正常增殖和分化，又称原癌基因，被致癌因素激活时，可诱导细胞癌变。,抑癌基因（tumorsuppressor gene，TSG）又称抗癌基因（anti-oncogene）、隐性癌基因（recessive oncogene），是正常细胞中存在的对原癌
8、基因表达功能进行调节的基因，可抑制细胞生长并能潜在抑制癌变。它的失活或突变具有促进肿瘤生长的潜势。抑癌基因活性的下降(功能缺失突变)也可引起癌症的发生。,含转录启动子,LTR,gag,pol,env,src,LTR,长末端重复序列,癌基因,正常的病毒基因,产生病毒表面糖蛋白,产生逆转录酶和整合酶,产生病毒 垮膜蛋白,产生p60src蛋白质，磷酸化蛋白。靶蛋白磷酸化,Rous于1910年首先发现鸡肉瘤病毒（后称劳斯肉瘤病毒RSV）,1966 诺贝尔奖,9.3 癌基因致癌的分子机制,病毒癌基因,纵向传播：原病毒随细胞分裂传递横向传播：原病毒转录表达（参与癌症的发生）,原癌基因致癌的分子机制,有时原
9、癌基因中一个核苷酸的突变会使基因表达异常的蛋白产物，从而使细胞向恶性转化。例如：膀胱癌EJ株C-Ha-ras基因外显子12密码子的突变,基因点突变,基因扩增,蛋白质结构未变化，但总量大大提高,基因在细胞中拷贝数增加，从而使可用的转录模板数大大增加。,染色体易位或重排,慢性粒细胞白血症(Chronic myeloid leukaemia，CML)is characterised bythe t(9;22)(q34;q11)reciprocal translocation,病毒启动子插入,基因间相互作用,肿瘤细胞中常常不是由一种C-onc表达异常，有时需几种C-onc发生改变，产生协同作用方可奏效
10、,正常细胞的基因，具有相对稳定的甲基化类型，特别是原癌基因的甲基化可使其难以表达。,原癌基因甲基化水平降低,Examples of Oncogenes,RAS-activated in many cancers(colon)c-MYC over-expressed in colon cancer(amplified in lung,rearranged in lymphoma)RET-MEN 2aMET-hereditary papillary renal cancerCDK4-familial melanomaBCR/ABL-chronic myelogenic leukaemiaBCL2-
11、follicular lymphoma,9.4 抑癌基因的作用及其致癌机制,抑癌基因在染色体中起稳定性作用；参与细胞分化以控制肿瘤形成，细胞的终末 分化可抑制细胞分裂；参与衰老过程，诱导细胞程序性死亡；通过调控细胞周期，调节细胞增殖。,正常情况下抑癌基因的作用：,抑癌作用通过抑瘤基因表达“抑癌蛋白”实现,抑癌基因改变的分子基础,错配修复基因缺陷,守门基因和看护基因的突变失活,守门基因（gatekeeper gene）是指在细胞恶性转化过程中与癌基因启动相关的基因。看护基因（caretaker，gene）是指保持细胞基因组稳定性，而与细胞恶性转化过程中癌基因的启动不直接相关的基因。,Geneti
12、c Control of Neoplastic Initiation and Promotion,Gatekeeper genesCaretaker genes,Examples of tumour suppressor genes includeRB1-retinoblastoma susceptibility gene WT1-Wilms tumour gene NF1-neurofibromatosis type 1 gene NF2-neurofibromatosis type 2 gene DCC-involved in colorectal cancer BRCA1,BRCA2-i
13、nvolved in breast cancer,Today we will look at these tumour suppressor genes:Rb(视网膜母细胞瘤)p53APC(腺瘤样结肠息肉),Knudsons“Two-Hit”Model for Retinoblastoma,Normal 2 intact copies,Predisposed 1 intact copy1 mutation,Affected Loss of both copies,p53 functions as a molecular node in the DNA-damage response,小 结,正
14、常细胞的生长与增殖是由两大类基因调控的正向调控信号：主要是起促进细胞生长和增殖，并且阻止其发生终末化倾向，癌基因起着这方面的作用。负向调控信号：主要使细胞成熟，促进终末分化，最后是细胞调亡，抑癌基因则在这方面起作用。正常情况下这两类信号保持着动态平衡，十分精确地调控细胞增殖和成熟。一旦这两类信号中有一类信号过强或过弱均会使细胞生长失控而恶变。,恶性肿瘤细胞在生物学研究中的应用,无限制增长特性 细胞分化幼稚 细胞寿命长 细胞的侵润性 致瘤性 细胞的遗传性,Normal,Tumour,Clonal selection of cancer cells,Variants over time,经典的抗原
15、递呈理论认为：外源性抗原经APC摄取后，在溶酶体中降解为短肽，由MHC类分子提呈给CD4+T细胞；内源性抗原在胞质溶胶中降解后，与MHC类分子结合，提呈给CD8+T细胞，激活细胞毒性T淋巴细胞(CTL)杀伤病毒感染细胞或肿瘤细胞。然而，通常情况下病毒感染或发生异常突变的细胞主要为实质细胞，它们大多不能迁移到淋巴器官与T细胞相互接触，同时也缺乏T细胞活化所必须的共刺激信号，故不能活化初始型CD8+T细胞。,外/内源性抗原示意图,1976年，Bevan首先发现交叉递呈(cross-presentation)的现象，随后，Sigal等也揭示外源性抗原能有效被APC的MHC类分子递呈。借助交叉递呈机制
16、，APC可以主动摄取病毒蛋白或肿瘤抗原，并通过MHC类分子将抗原呈递给CD8+T细胞，从而产生有效的CTL应答，达到清除靶细胞的目的。近年来更多研究表明，凋亡或坏死的细胞、热休克蛋白、病毒蛋白、颗粒性抗原，甚至可溶性抗原等都可以通过交叉递呈途径活化CTL应答。交叉递呈在机体抗病毒及其它病原体感染和肿瘤免疫等方面起着非常重要的作用。,树突状细胞(DC)、巨噬细胞等专职APC都具有交叉活化CD8+T细胞的能力，而DC是体内最有效的专职APC，抗原摄取能力强且能活化初始型T细胞，是执行交叉递呈功能的主要细胞。DC需要经历由不成熟向成熟阶段的转变才能有效刺激T细胞活化。不成熟DC捕获抗原后，在炎性因子
17、的刺激下分化为成熟DC，细胞的抗原摄取能力下降，而抗原递呈及刺激T细胞活化的能力增强。此过程同时伴随细胞表面MHC类分子、类分子、共刺激分子CD80、CD86以及T细胞黏附分子CD48、CD58等的上调表达。此外，DC还通过延伸其树突状突起，增加与T细胞相互作用的机会。,流式细胞术、激光共聚焦等新技术的应用为研究抗原交叉递呈的分子机制提供了保障；近年研究发现并非所有的DC亚型都能参与交叉递呈，只有表达CD24,CD8和CD103 分子的DC才参与细胞交叉递呈；TLR具有激活DCs，增强其抗原交叉递呈的能力。,The molecular mechanisms involved in classi
18、cal cross-priming are illustrated.Dendritic cells(DCs)take up antigen by distinct endocytosis mechanisms and present it to CD4+T helper(TH)cells through MHC class II molecules and cross-present it to CD8+cytotoxic T lymphocytes(CTLs)through MHC class I molecules.Activated CD4+TH cells can stimulate
19、CTLs through the production of interleukin-2(IL-2)and license DCs for cross-priming through CD40 ligand(CD40L)CD40 interactions.Licensed DCs upregulate expression of co-stimulatory molecules,such as CD70,CD80 and CD86,and downregulate inhibitory molecules,such as programmed cell death ligand(PDL1).T
20、oll-like receptor(TLR)ligands further activate DCs and increase their cross-presentation activity.Cross-primed CTLs are programmed for survival and cease TNF-related apoptosis-inducing ligand(TRAIL)production.Helpless CTLs activated by unlicensed DCs die following secondary encounter with antigen in
21、 their effector phase,Chemokine-mediated regulation of cross-priming is illustrated.CD4+TH cells,and the DCs they license,produce CC-chemokine ligand 3(CCL3),CCL4 and CCL5 in the presence of TLR ligands,which recruit naive CTLs for classical cross-priming.Alternatively,DCs licensed by natural killer
22、 T(NKT)cells produce the CC-chemokine receptor(CCR4)ligand CCL17,and NKT cells themselves produce the CCR4 ligand CCL22,which recruit naive CCR4+CTLs for cross-priming.The CCR4-and CCR5-mediated recruitment pathways are synergistic.In this figure,dashed arrows indicate antigen routing for crossprese
23、ntation.-GalCer,-galactosylceramide;TCR,T cell receptor.,Recruitment of cross-primed effector cytotoxic T lymphocytes into non-lymphoid tissues.Viral infection of tissue cells leads to their secretion of pro-inflammatory cytokines and interferons that upregulate the expression of adhesion molecules,
24、such as intercellular adhesion molecule 1(ICAM1),by endothelial cells.Effector cytotoxic T lymphocytes(CTLs)attach to these molecules and are nonspecifically recruited from the bloodstream into non-lymphoid tissues.In addition,recent studies have revealed two antigen-specific recruitment mechanisms:
25、first,endothelial cells in certain tissues,such as the liver,pancreatic islets or the brain,can cross-present microbial antigen,which allows them to selectively recruit antigen-specific CTLs.Second,when specific CD4+T helper(TH)cells detect microbial(or self)antigen on non-cross-presenting tissue DC
26、s,ligands for CC-chemokine receptor 5(CCR5)or CXC-chemokine receptor 3(CXCR3)are produced that recruit CTLs into the infected tissue.LFA1,lymphocyte function-associated antigen 1;TCR,T cell receptor.,Growth factors(I)Growth factor receptors(II)Signal-transduction proteins(III)Transcription factors(I
27、V)Pro-or anti-apoptotic proteins(V)Cell cycle control proteins(VI)DNA repair proteins(VII).Mutations in I-IV generally give rise to oncogenes.Class VI proteins act as tumour suppressors;mutations in these act recessively to release cells from control and surveillance,increasing the probability of cancer developing.,7 proteins controlling cell growth,
