细胞与细胞工程课件.ppt

单击此处编辑母版标题样式,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,*,单击此处编辑母版标题样式,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,*,Cell&Cell Engineering,School of Life Sciences,Xiamen University,Dr.Gaoliang Ouyang(,欧阳高亮,),Dr.Shuyong Lin(,林舒勇,),1,2012-2013年第2学期 生命科学导论安排,讲座号,题目,任课教师,第一讲,细胞与细胞工程,欧阳高亮，林舒勇,第二讲,基因与基因工程,徐虹，顾颖,第三讲,微生物与传染性疾病,陈毅歆，陈航姿王彦海,第四讲,营养与健康,金利华，周克夫,第五讲,人类遗传学与优生,许晔，李光,第六讲,生物多样性与环境保护,郑文教，杨盛昌,第七讲,生物技术在农业和医学领域的应用,吴婷，程喆,第八讲,生物伦理与社会决策,张赛群，陈长平,课程召集人：罗大民,13959249809,dmluo,2,Fertilized egg can be developed into embryo and used to isolate embryonic stem cells,3,生命科学研究的范畴和层次,核心领域,：生物学,（动物学，植物学等），,生物医学科学,（疾病相关研究）,小分子,-,生物大分子,-,细胞器,-细胞,-,组织,-,器官,-,生物个体,-,群落,-,生态系统,4,Science,Dec.2012,Science 2012,年度十大科学进展,5,心肌细胞,肝细胞,胰腺细胞,精子,卵子,胚胎,干细胞,6,饮食对肠道微生物组产生的作用，以及它们对患病风险的影响,癌症免疫学研究,黑色素瘤治疗方案（,dabrafenib,（,BRAF,抑制剂）和,trametinib,（,MEK,抑制剂）组合药物）,干细胞试验的基础研究和临床研究,单细胞测序技术,人类连接组项目,(Human Connectome Project),计划,植物学基础研究,调控植物生长的分子遗传学机制,Science,和,Nature 2013,年度生命科学领域热点预测,7,Nature,杂志则指出今年干细胞试验的基础研究和临床研究也许会取得新进展，其编辑认为一项利用人类胚胎干细胞（,hESC,）进行的早期临床试验应该在,2013,年会获得具有里程碑意义的研究结果，这个由著名的先进细胞技术公司进行的实验，正在将源自,hESC,的视网膜细胞注入,36,名患有两种形式的无法治愈的退行性失明的患者眼中。这是目前唯一一家获得美国食品与药物管理局（,FDA,）批准开展,hESC,治疗试验的公司，并且先进细胞技术公司希望,FDA,能够为其开绿灯，从而于今年在病人中对诱导自成熟细胞的干细胞进行测试。,8,细胞工程,细胞工程是运用细胞分子生物学理论与技术，定向改造细胞乃至生物。,用这种技术可以对细胞进行操作，达到定向改造生物的目的,是人们根据科学设计改变细胞的遗传基础，通过无菌操作，大量培养所需细胞，诱导形成组织甚至发育为完整个体的技术。,迄今为止，人们已经从基因水平、细胞器水平以及细胞水平开展了多层次的大量工作，在细胞培养、细胞融合、细胞代谢物的生产和生物克隆等诸多领域取得一系列令人瞩目的成果，其中代表细胞水平的细胞工程研究也显示其巨大的应用前景。,9,第一节 细胞的特征,细胞的基础知识,细胞的生长、分化与死亡,第二节 动物细胞工程,动物细胞培养,单克隆抗体,动物克隆,干细胞工程,10,第一节 细胞的特征,细胞,是生命的,结构,和,功能,的基本单位。,细胞是生命活动的基本单位。,单细胞生物，一个细胞就是一个个体，而多细胞生物则由许多细胞成一个个体。我们所看到的生物体都是由多细胞所组成。在多细胞生物体中，由形态结构相似的细胞再组成组织，执行着生物体中的部分功能。,11,第一节 细胞的特征,细胞的基础知识,细胞的生长、分化与死亡,第二节 动物细胞工程,动物细胞培养,单克隆抗体,动物克隆,干细胞工程,12,根据细胞的内部结构，可将生物界的细胞分为两大类：,原核细胞（,prokaryotes,）,和,真核细胞（,eukaryotes,）,细菌、蓝藻和放线菌等由原核细胞构成的有机体称为,原核生物,，几乎所有的原核生物都由单个原核细胞构成。,由真核细胞构成的有机体则称为,真核生物，,真核细胞大多存在于多细胞生物中。,单细胞生物与多细胞生物,在适应环境等方面各有千秋,.,（一）细胞分类,一,.,细胞的基础知识,13,（二）细胞的大小和形态,支原体（,mycoplasma,）,巨型乌贼神经细胞,（,nerve cell,）,蕨藻（,caulerpa,）,14,细胞形态的多样性,硅藻,草履虫,15,无论各种细胞的大小和形态有多大的差异，它们都由一层具有一定生物学功能的,细胞膜,包裹在细胞外层。由于这层细胞膜将细胞内部和外部环境分隔开，使细胞内部的各种生命活动发生在特定的环境中保障了单细胞个体的生命活动和多细胞个体中不同功能细胞的相对独立性，使其成为生物体的功能单位。,（三）细胞的结构,16,结构简单，因而相对较小,没有细胞核和大多数细胞器（恒定的内膜系统,),细菌和古细菌两类,三个结构区域：鞭毛或纤毛，胞外被膜和细胞质,I.,原核细胞,Prokaryotic cells,17,三大结构体系：,生物膜系统,：质膜、内膜系统（细胞器）,遗传信息表达系统,：染色质,(,体,),、核糖体、,mRNA,、,tRNA,等,细胞骨架系统,：胞质骨架、核骨架,II.,真核细胞（,eukaryotic cells,）,chromatid,18,具有核膜和由膜围成的各种,细胞器,，如内质网、高尔基体、线粒体、叶绿体、溶酶体等。,内膜系统将细胞质分隔成不同的区域，即所谓的,区隔化,(compartmentalization),。区隔化使细胞内表面积增加了数十倍，代谢能力增强。,19,第一节 细胞的特征,细胞的基础知识,细胞的生长、分化与死亡,第二节 动物细胞工程,动物细胞培养,单克隆抗体,动物克隆,干细胞工程,20,G,0,期,:,休眠期,G,1,期,:DNA,合成前期,S,期,:DNA,合成期,G,2,期,:,DNA,合成后期,M,期,:,有丝分裂期,（一）细胞增殖,二,.,细胞的功能,20 h,细胞分裂三种方式,21,(,二,),细胞分化,多细胞生物体是由各种各样形态和功能都不同的细胞群所组成。高等动、植物由受精卵细胞开始的胚胎发生过程随着细胞分裂次数的增加而使得早期胚胎的细胞数量也增加许多。其中有些细胞在形态、结构和功能上逐渐发生了差异，这种细胞之间差异的发生过程就是细胞分化,。,个体发育的过程就是细胞分化的过程，个体的各种器官和组织都是通过细胞分化形成的。,囊胚,原肠胚,22,Examples of names and types of cell death.,(A,I)Electron microscopy;(J,L)phase contrast.(A)Normal HeLa cells;(B)treated with staurosporin to induce apoptosis;orC)treated with thapsigargin to induce autophagy(Galluzzi,et al,2007).,(D)Apoptotic,and(E)necroptotic Jurkat cells(Degterev,et al,2005).,(F)Paraptotic 293T cells(Sperandio,et al,2000;copyright(2000)National Academy of Sciences,USA).,(G)Necrotic and(H)autophagic iBMK cells(Degenhardt,et al,2006;Mathew,et al,2007).,(I)Ras-expressing U251 glioblastoma cells showing macropinosomes(J.H.Overmeyer,A.Kaul,E.E.Johnson&W.A.Maltese,unpublished data).(J),Dictyostelium,cells,vegetative,(K)undergoing vacuolar autophagic cell death,and(L)undergoing necrotic cell death(Laporte,et al,2007).,凋亡,自吞噬,凋亡,坏死,自吞噬,坏死凋亡（,apoptosis,，程序性死亡），坏死（,necrosis,），自吞噬（,autophagy,），衰老（,senescence,）,(,三,),细胞衰老和死亡,23,Hayflicks Limit:human cells derived from embryonic tissues can only divide a finite number of times(50)in culture,Fibroblast,Division,20 times,50 times,细胞衰老,24,Limitless Replicative Potential,Human somatic cells have a finite replicative potential,once cells progressed through a certain number of doublings,cells will stop growing-a process termed,senescence,.,25,Loss of proliferative capacity with age,26,Apoptosis and normal morphogenesis(Mouse paw),27,细胞死亡控制失效的后果：,该死的不死：肿瘤,不该死的死了：退行性疾病（,AD,，,Parkinson,s Disease,）,28,Tumors as Complex Tissues,The tumor microenvironment is regulated not only by cancer cells but also by endothelial cells,immune cells and stromal elements,-Hanahan D,Weinberg RA.,Cell,2000.,(,四,),肿瘤,29,Cell cycle checkpoint,30,Tumor invasion-metastasis cascade,（肿瘤侵袭转移）,31,Cancers Result From genetic Mutation,Alteration in the cells DNA sequence,Epigenetic change:a change in the pattern of gene,expression without a change in the DNA sequence,Chemical carcinogens,Ionizing radiations,Viruses,Agents that give rise to cancer,32,Many,but not all,cancer-causing agents damage DNA,Fungal toxin aflatoxin B1,The more potent ones are relatively inert chemically and become damaging only after they have been changed to a more reactive form by metabolic processes,Metabolic activation of a carcinogen,33,Zhou BB,et al,.,Nat Rev Drug Discov,.2009.,Cancer stem cells(CSCs),also known as tumor initiating cells or tumorigenic cells,refer to a small subset of tumor cells that have stem-like cell properties,the ability to self-renew,differentiate into heterogeneous populations of cancer cells,and seed new tumors in a xenotransplant system.,Cancer stem cell(,肿瘤干细胞）,34,第一节 细胞的特征,细胞的基础知识,细胞的生长、分化与死亡,第二节 动物细胞工程,动物细胞培养,单克隆抗体,动物克隆,干细胞工程,35,体外培养细胞的基本类型,形态：,成纤维样,细胞和,上皮样,细胞,根据细胞能否在支持物上生长，分为,贴附型,和,悬浮型,。,成纤维样细胞,上皮样细胞,悬浮型细胞,第二节 动物细胞工程,一、动物细胞培养,36,细胞培养的基本设备,无菌室,无菌操作台,37,二氧化碳培养箱,倒置显微镜,细胞计数仪,38,透射电子显微镜,激光扫描共聚焦显微镜,39,培养瓶,培养皿,培养板,培养液,40,第一节 细胞的特征,细胞的基础知识,细胞的生长、分化与死亡,第二节 动物细胞工程,动物细胞培养,单克隆抗体,动物克隆,干细胞工程,41,二,.,单克隆抗体,1975,年英国科学家,Milstein,和,Kohler,将产生抗体,的淋巴细胞同肿瘤细胞融合，成功建立了单克隆抗,体技术，而获得,1984,年诺贝尔医学和生理学奖。,每个,B,淋巴细胞仅专一地产生、分泌一种针对某,种抗原决定簇的特异性抗体，而肿瘤细胞可以无限,增殖，因此杂交瘤细胞可在体外培养或移植到体内,条件下分泌大量单克隆抗体。,单克隆抗体技术的最主要优点是可以用不纯的抗,原分子大量制备纯一的单克隆抗体。,42,43,单克隆抗体的应用,分子细胞生物学基础研究,疾病的定向治疗（肿瘤免疫疗法，风湿性关节炎等）,疾病的早期诊断,超过,150,亿美元的市场,44,第一节 细胞的特征,细胞的基础知识,细胞的生长、分化与死亡,第二节 动物细胞工程,动物细胞培养,单克隆抗体,动物克隆,干细胞工程,45,三,.,动物克隆,生物学上，克隆指的是低等生物无性繁殖产生遗传信息完全一致的群体的自然存在的过程。,生物技术领域，克隆指的是复制（拷贝）,DNA,片段（分子克隆），细胞（细胞克隆，应用于干细胞）乃至生物体（生殖克隆）的手段。,生殖克隆一般指创造遗传特性上完全一致的多细胞个体的过程，是一种无性繁殖。,人为创造,：,体细胞核移植,，利用显微外科技术，将一个体细胞核移植到一个去核的卵细胞中，再植入母体子宫，便可不经授精而发育。,胚胎分裂（胚胎孪生）,自然存在：个别雌性昆虫，甲壳类和蜥蜴,成熟个体的复制,46,克隆研究的历史及现状,Hans spemann,1869-1941,1928,年进行了首次核移植实验,1935,年 获诺贝尔生理医学奖,1938,年预言了克隆高级生物的可能性,德国胚胎生物学家（,the father of cloning,）,47,Hans Spemann,的蝾螈核移植实验,1938,年,Spemann,和他的学生发现，把发育早期的蝾螈细胞核移植到去除了细胞核的发育晚期蝾螈胚胎中，胚胎细胞可以继续发育成为一个完整的蝾螈。,48,49,1952,年,Briggs,和,King,用囊胚细胞核克隆了蝌蚪,Robert Briggs,(1911-1983),Thomas King,(1921-2000).,紫外线照射,未受精卵,移核卵,蝌蚪,197,移核卵，,104,个分裂发育，,35,各发育为胚胎，,27,个成为蝌蚪,The first cloned vertebrate,囊胚细胞,分化早期的胚胎细胞,50,1962,年,John Gurdon,用肠细胞（,分化终末细胞,）核克隆青蛙,John Gurdon,(1933-),紫外线照射,未受精卵,移核卵,蝌蚪,提取肠上皮细胞核,注入细胞核,蝌蚪,囊胚,囊胚,不分裂,畸形胚,成蛙,细胞可以“重编程”,克隆技术的基础,51,1979年,Karl Illmensee,宣布运用核移植法成功克隆,了3只小鼠，但该结果没有得到科学界公认,198,6,年丹麦科学家,Steen Willadsen,成功利用,胚胎,细胞,克隆出一头绵羊，这是首次证实的通过核移植技,术克隆哺乳动物,哺乳动物的克隆,1995年7月 苏格兰,Roslin,研究所的,Ian Wilmut,和,Keith,Campbell,运用,早期分化的胚胎细胞,克隆出了两头绵羊，,命名为,Megan,和,Morag,这被看作是多莉诞生的前奏,1996年7月5日第一个用,成年体,乳腺,细胞,克隆产生的哺乳动物绵羊多莉（,Dolly）,诞生,52,July 5,1996 February 14,2003,多莉（,Dolly,）,Royal Museum in Edinburgh,53,Wilmut,A.E.Schnieke,J.McWhir,A.J.Kind and K.H.Campbell.Viable offspring derived from fetal and adult mammalian cells.,Nature,1997,385:810813.,54,克隆羊多莉引发的动物克隆热,牛，,05/07/1998,日本,猴，,14/01/2001,美国,猫，,14/02/2002,美国,骡，,29/05/2003,美国,小鼠，,05/12/1999,美国,骆驼，,8/04/2009,阿联酋,55,(1),遗传素质完全一致的克隆动物将更有利于开展对动物,(,人,),生长、发育、衰老和健康等机理的研究,；,(2),有利于大量培养,品质优良的家畜,;,(3),经,转基因的克隆哺乳动物,，将能为人类提供源源不断的廉价的药品、保健品以及较易被人体接受的移植器官；,(4),科学家将很快地从目前的同种克隆技术推进到,异种克隆,即借腹怀胎的新领域,这无疑将大大促进对濒临灭种的哺乳动物的保护工作。,克隆技术将对,21,世纪产生的重大影响（？）,56,第一节 细胞的特征,细胞的基础知识,细胞的生长、分化与死亡,第二节 动物细胞工程,动物细胞培养,单克隆抗体,动物克隆,干细胞工程,57,Stem cells are powerful tools in biology and medicine.What can scientists do with these cells to exploit their incredible potential?,Adapted fromPrinciples of Biologypublished by,Nature Education.,JAMES KING-HOLMES/SCIENCE PHOTO LIBRARY,A mouse embryo is held in place during nuclear transfer.The eggs genetic material has been removed and an adult cell nucleus is injected in its place.,四,.,干细胞工程,58,Pluripotent,:can transform into any one of the 220 cell types in the human body.,Self-renewal,:one stem cell can also divide to produce millions more stem cells.,1.What are Stem Cells?,59,in nearly all animals,from tiny worms to mice and humans.,embryonic stem cells,adult stem cells,induced pluripotent stem(iPS)cells,2.Types of Stem Cells,60,Embyonic Stem Cells,VS.,Adult Stem Cells,Stem Cells:Flexible friends,Nature,483,S22S26,61,In the past decade,scientists have also learned to make stem cells from regular mature,differentiated cells.These cells are called induced pluripotent stem cells because scientists,force,them to become pluripotent even after they have reached a,differentiated,state.,By turning up the expression of just a few genes,scientists can force a skin cell,for example,to retrace its developmental pathway backwards all the way to a flexible pluripotent state.,Induced Pluripotent Stem Cells,62,3.Medical Applications,Stem cells can teach us about the biology of pluripotent cells,how the human body develops,and what happens during disease.,They are also a tool for developing safer ways to,design and test new medications,.,they are the basis for,regenerative medicine,using stem cells to repair damaged tissue.,63,The discoveries of Gurdon and Yamanaka have shown that specialised cells can turn back the developmental clock under certain circumstances.Although their genome undergoes modifications during development,these modifications are not irreversible.We have obtained a new view of the development of cells and organisms.,Research during recent years has shown that iPS cells can give rise to all the different cell types of the body.These discoveries have also provided new tools for scientists around the world and led to remarkable progress in many areas of medicine.iPS cells can also be prepared from human cells.,For instance,skin cells can be obtained from patients with various diseases,reprogrammed,and examined in the laboratory to determine how they differ from cells of healthy individuals.Such cells constitute invaluable tools for understanding disease mechanisms and so provide new opportunities to develop medical therapies.,From surprising discovery to medical use,皮肤细胞,iPS,神经细胞,心肌细胞,肝细胞,精子、卵子,64,Why is a therapeutic cloning approach important?,By growing new body parts from existing ones from the same person we can avoid,transplant rejection problems,.,By using cells re-engineered or“cloned”from ourselves we can become our own,transplant source,-similar to banking blood for ourselves before a surgical operation.,65,People in the US affected by diseases that may be helped by stem cell research,ConditionNumber of Persons Affected,Cardiovascular diseases 58 Million,Autoimmune diseases 30 Million,Diabetes16 Million,Osteoporosis10 Million,Cancer8.2 Million,Alzheimers disease4 Million,Parkinsons disease1.5 Million,Burns(severe)0.3 Million,Spinal cord injuries0.25 Million,Birth defects150,000(per year),Total 128.4 Million,-,Data from the Patients Coalition for Urgent Research,Washington,DC(according to Perry,Ref.267).,66,NATURE,|NEWS,Chinas stem-cell rules go unheeded,Health ministrys attempt at regulation has had little effect.,Nature,484,149150(12 April 2012),Its boom time for firms selling stem-cell treatments,such as those derived from umbilical blood.,LIU HAIFENG/XINHUA PRESS/CORBIS,67,NATURE,|NEWS,Stem-cell therapy takes off in Texas,A boom in unproven procedures is worrying scientists.,Nature,483,1314(01 March 2012),Celltex hosts the largest stem-cell bank in the United States.,TYLER RUDICK,68,4.Development of the Stem Cell technique,Source:are the Stem Cells?,自然胚胎,克隆人胚胎,-,核移植,/,混种胚胎,-,核移植,体细胞诱导多能干细胞,(,体细胞转分化,),70,In,1868,German biologist Ernst Haeckel first used the term stem cell to describe a zygote.,Researchers have been working with,mouse,embryonic stem cells since,1981.,the field took off in,1998,when researchers isolated,human,embryonic stem cells for the first time.,（最早的人类多能干细胞是从体外受精的临床病例中得来的多余胚泡。这个里程碑式的事件是,James Thomson,领导的,University of Wisconsin,Madison,的实验室在,1998,年的成果。）,3.1 Isolation and culture of Stem Cells,71,3.2 Objections to Stem Cell Reasearch,72,2008,纽卡斯尔大学将从,人类皮肤细胞,中提取的,DNA,注入来自,母牛卵巢的卵子,，卵子的所有遗传物质基本上都被去除。,3.3 Interspecies Embryos,2003,上海第二医科大学盛慧珍教授研究组将,人的体细胞核,植入去核的,兔卵细胞,中。,73,3.4 Adult Stem Cells,do not raise the,ethical questions,that embryonic stem cells do because no embryos are needed,exist in,very,small numbers,often,buried deep in the tissue,mainly used for two treatments:,bone-marrow transplants,to rebuild a patients immune system,and,skin grafts,to replace skin over a burn or other injury,other applications for adult stem cells,？,use drugs to activate a patients own adult stem cells to perform the necessary repairs,？,74,For a long time,scientists thought that once a cell differentiated,it could never regain its pluripotency.They assumed that it must be set in its identity because it had so many genes turned on and off in ways particular to that cell type.,In,2006,however,Kazutoshi Takahashi and Shinya,Yamanaka,of Kyoto University in Japan proved otherwise.They turned,mouse skin cells,specifically,fibroblasts into stem cells.These were the first induced pluripotent stem(iPS)cells.,3.5 Induced Pluripotent Stem Cells,75,The first direct reprogramming of adult human fibroblasts.,Ian Wilmut,Scottish Centre for Regenerative Medicine,University of Edinburgh,In 2007 issues of,Cell,and,Science,Yamanaka and colleagues(Takahashi et al.,2007)and Yu and colleagues(Yu et al.,2007)demonstrate that expression of,four specific transcription factors,gives,adult human fibroblasts,many of the characteristics of human embryonic stem cells.Refinements of this procedure will make it possible to,produce pluripotent human cell lines without use of an embryo,.There are profound scientific and social implications of this research.,Cell Stem Cell,2007 Dec 13;1(6):593-4.,I decided a few weeks ago not to pursue nuclear transfer-the method used to create Dolly the sheep,he said,admitting the new method was easier to accept socially.,76,十年科学进展,1.The Dark Genome,：基因组中的“暗物质”,3.Ancient DNA,：古生物的,DNA,5.Reprogramming Cells,：细胞重编程,6.The Microbiome：,人体内微生物组,8.Inflammation,：炎症反应的阴暗面,Science,Dec.2010,77,The Nobel Prize in Physiology or Medicine 2012.Nobelprize.org.21 Feb 2013 www.nobelprize.org/nobel_prizes/medicine/laureates/2012/,The Nobel Prize recognizes two scientists who discovered that mature,specialised cells can be reprogrammed to become immature cells capable of developing into all tissues of the body.Their findings have revolutionised our understanding of how cells and organisms develop.,78,John B.Gurdon discovered in 1962 that the specialisation of cells is reversible.The DNA of the mature cell still had all the information needed to develop all cells in the frog.,Would it e