《动植物细胞工程》PPT课件.ppt

4 动物细胞与组织培养 4 1动物细胞特点 无细胞壁倍增时间长 生长缓慢需氧量少 对搅拌敏感聚集体形成原代细胞培养50代即开始退化 4 2动物细胞培养定义 动物细胞与组织培养是从动物体内取出细胞或者组织 模拟体内的生理环境 在无菌 适温和丰富的营养条件下 使离体细胞或者组织生存 生长并维持结构和功能的一门技术 是动物细胞工程的基础 类型 1 细胞培养 2 组织培养 3器官培养 4 3发展历史 1885年WRoux鸡胚神经板1907 美国的哈里森使用盖玻片悬滴培养蛙胚神经组织细胞ACarrel无菌技术1951WEarle培养基 4 4生长特性 贴附生长型如人胚肺细胞 Hela细胞 巨噬细胞 神经细胞上皮样 成纤维样 无定形单层 汇合悬浮生长型细胞如血液白细胞 淋巴组织细胞等 Figure4 2Thefibroblast A Phase contrastmicrographoffibroblastsinculture B Drawingsofalivingfibroblastlikecellinthetransparenttailofatadpole showingthechangesinitsshapeandpositiononsuccessivedays Notethatwhilefibroblastsflattenoutinculture theycanhavemorecomplex process bearingmorphologiesintissues A courtesyofDanielZicha B redrawnfromE Clark Am J Anat 13 351 379 1912 常用术语 Cell tissue organculture贴壁依赖性 汇合原 初代培养Primaryculture传代细胞系 有限 无限 连续 已建成的 二倍体 转化细胞株 有限 无限 连续 克隆接触抑制 4 5体外培养细胞基本技术 体外培养特点体外培养工具体外培养条件体外细胞生长增殖过程培养方法大规模培养技术 4 5 1体外培养特点 营养条件苛刻适应性差 敏感培养时间长 易污染 Animalcellsaremoredifficulttoculturethanmicroorganismsbecausetheyrequiremanymorenutrientsandtypicallygrowonlywhenattachedtospeciallycoatedsurfaces Despitethesedifficulties varioustypesofanimalcells includingbothundifferentiatedanddifferentiatedones canbeculturedsuccessfully 4 5 2体外培养工具 空心纤维微球 Cells tissuesgrowninculturelongperiodoftimeBASICSTERILETECHNIQUE Workenvironmentandsurface Plasticwareandglassware Handlingtechniques Sterilizationofsolutionsformaintenancegrowthortreatment WORKENVIRONMENTANDSURFACELaminarflowhoodRelativelyenclosedspaceLittletrafficflowingpastworkspaceConfinedspacecanbeeasilycleanedandmaintainedMusthaveannualcheckupofHEPAfiltersShouldhaveinternaloutletsforelectricity vacuumandgasFrequentlyoutfittedwithUVlightPreventcontaminationbydailyscrubwith70 ethanolUseclosedflaskattachedtovacuumforspentmediumSeparatesterileroomAllincomingaircirculatedthroughHEPAfilters PLASTICWAREANDGLASSWAREPipettes bottles flasks petridishes Plastic betterattachmentandcellgrowthofmonolayercellculture moreexpensivethanreusableglass suitableforstorageat4 C Glass Canwithstandtemperatures 0 Cuseformaintainingfrozenstocks Steamsterilizebyautoclaving reliesonsteampressureandhighheat usefast drycycletodrycondensate use wet cycleforsaltsolutionstopreventevaporation looselyplacecapsonbottles 4 5 3体外培养条件 温度 37度pH 7 2 7 4气体 氧 二氧化碳 氮气营养条件 天然 合成培养基 需要多种氨基酸 维生素 辅酶 核酸 嘌呤 嘧啶 激素和生长因子 其中多种成分可以由血清提供低 无血清培养基贴壁因子 生长因子多为肽激素 有胰岛素 表皮生长因素 EGF 成纤细胞生长因素 fibroblastgfowthfactor FGF 血小板来源增殖因素 platelet derivedgrowthfactor PDGF 以及生长激素释放抑制因子 somatostatin SRIH RichMediaAreRequiredforCultureofAnimalCells Nineaminoacids referredtoastheessentialaminoacids cannotbesynthesizedbyadultvertebrateanimalsandthusmustbeobtainedfromtheirdiet Animalcellsgrowninculturealsomustbesuppliedwiththesenineaminoacids namely histidine isoleucine leucine lysine methionine phenylalanine threonine tryptophan andvaline addition mostculturedcellsrequirecysteine glutamine andtyrosine Theotheressentialcomponentsofamediumforculturinganimalcellsarevitamins whichthecellscannotmakeatallorinadequateamounts varioussalts glucose andserum thenoncellularpartoftheblood MostCulturedAnimalCellsGrowOnlyonSpecialSolidSurfaces Theextracellularmatricesinvariousanimaltissuesconsistofseveralcommoncomponents fibrouscollagenproteins hyaluronan orhyaluronicacid alargemucopolysaccharide andcovalentlylinkedpolysaccharidesandproteinsintheformofproteoglycans mostlycarbohydrate andglycoproteins mostlyprotein 4 5 4体外细胞生长增殖过程 原代培养期传代培养期衰退期 细胞系建立与鉴定 基本概念细胞系档案的有关说明来源生物学特性培养条件和方法鉴定 纯度 稳定性 细胞学特征 污染检查 4 5 5培养方法 悬滴旋转管灌注小室培养瓶培养板 细胞克隆 克隆形成率技术稀释铺板法饲养层法琼脂法分离技术 克隆化环 辐射 挑选法 细胞周期同步化 4 5 6大规模培养技术 悬液培养 使用微载体技术多孔微载体 不仅适合贴壁细胞 也适合悬浮细胞的固定化连续灌流培养微囊 半透膜 化培养 海藻酸钠ALG 多聚赖氨酸PLL中空纤维 4 5 7动物细胞培养应用 1962年开始 用于生物医学研究 生产酶制剂 生长因子 疫苗 单抗等 Vero细胞和狂犬病毒的培养工艺 DMEM培养基 胎牛血清 其他成分 锥形瓶逐级放大培养 加碱 碳酸氢钠 加糖 葡萄糖 灌注培养液 微载体 5L种子罐培养 培养液 50L生物反应器 接种狂犬病毒 出液口 收获病毒 4 6 组织工程 组织工程组织培养上皮组织肌肉组织神经组织 组织工程 TissueEngineering 是近年来正在兴起的一门新兴学科 组织工程一词最早是由美国国家科学基金会1987年正式提出和确定的 它是应用生命科学和工程学的原理与技术 在正确认识哺乳动物的正常及病理两种状态下结构与功能关系的基础上 研究 开发用于修复 维护 促进人体各种组织或器官损伤后的功能和形态生物替代物的科学 组织工程的核心就是建立细胞与生物材料的三维空间复合体 即具有生命力的活体组织 用以对病损组织进行形态 结构和功能的重建并达到永久性替代 共基本原理和方法是将体外培养扩增的正常组织细胞 吸附于一种生物相容性良好并可被机体吸收的生物材料上形成复合物 将细胞 生物材料复合物植入机体组织 器官的病损病分 细胞在生物材料逐渐被机体降解吸收的过程中形成新的在形态和功能方面与相应器官 组织相一致的组织 而达到修复创伤和重建功能的目的 生物相容性好 可被人体降解吸收的组织工程支架材料称为细胞外基质 ECM 其功能是为细胞提供生存空间 使细胞获足够的营养物质 进行气体交换 并使细胞按预制形态的三维支架生长 在细胞和生物材料的复合体植入机体病损部位后 生物支架被降解吸收 但种植的细胞继续增殖繁殖 形成新的具有原来特殊功能和形态的相应组织器官 种子细胞 自体 同种异体 异种组织细胞等细胞外基质 extracelluarmatrix ECM 理想的ECM应具有以下特点 生物相容性好 在体内不引起炎症反应和毒性反应 有可吸收性 能彻底地被自身组织所取代 有可塑性 可塑为任意的三维结构 植入后在体内仍可保持特定形状 表面化学特性和表面微结构利于细胞的粘附和生长 降解速率可根据不同细胞的组织再生速率而进行调整 天然 胶原人工 聚乳酸 polylacticacidPLA 聚羟基乙酸 polyglycolicacidPGA 两者的共聚物 PGA PLA 聚 羟基丁酯 PHB 聚乳酸 已内酯的共聚物 PLC 聚原酸酯 聚磷本酯 聚酸酐等 组织工程临床应用 组织工程中临床应用是在组织构建完成了动物试验之后 在人体上的应用 这也是组织工程的最后一步 目前 组织工程的研究只有活性皮肤达到了这一步 Figure22 1Mammalianskin A Schematicdiagramsshowingthecellulararchitectureofthickskin B Photographofacross sectionthroughthesoleofahumanfoot stainedwithhematoxylinandeosin Theskincanbeviewedasalargeorgancomposedoftwomaintissues epithelialtissue theepidermis whichliesoutermost andconnectivetissue whichconsistsofthetoughdermis fromwhichleatherismade andtheunderlyingfattyhypodermis Eachtissueiscomposedofavarietyofcelltypes Thedermisandhypodermisarerichlysuppliedwithbloodvesselsandnerves Somenervefibersextendalsointotheepidermis Figure22 19Cross sectionofmammalianepidermis A Schematicdiagram B Photomicrographofasectionthroughthesoleofthefoot hematoxylinandVanGiesonstain Thegranularcellsbetweenthepricklecellsandtheflattenedsquamesareinthepenultimatestagesofkeratinization theyappeargranularbecausetheycontaindarklystainingaggregatesofamaterialcalledkeratohyalin whichisthoughttobeinvolvedintheintracellularcompactionandcross linkingofthekeratin Keratohyalinconsistsmainlyofaproteinknownasfilaggrin Inadditiontothecellsdestinedforkeratinization thedeeplayersoftheepidermisincludesmallnumbersofcellsofdifferentcharacter notshownhere includingmacrophagelikeLangerhanscells derivedfrombonemarrow melanocytes derivedfromtheneuralcrest andMerkelcells whichareassociatedwithnerveendingsintheepidermis SeealsoFigure22 1 Figure22 21Thecolumnarorganizationofsquamesintheepidermallayerofthinskin Thestructureisrevealedbyswellingthekeratinizedsquamesinasolutioncontainingsodiumhydroxide Thistypeoforganizationoccursonlywheretheepidermisisthin Somestudiessuggestthateachsuchcolumnisa proliferativeunit correspondingtoasinglestemcellamongthe10 12basalcellsonwhichthecolumnrests Figure22 22Animmortalstemcell Eachself renewingpatchofepidermismustcontainineachcellgenerationatleastone immortal stemcell whosedescendantswillstillbepresentinthepatchinthedistantfuture Thearrowsindicatelinesofdescent Animmortalstemcellisshownhereoccupyingthesamepositionineachcellgeneration Otherbasalcellsmightbebornchemicallydifferentinawaythatcommitsthemtoleavethebasallayeranddifferentiate ortheytoomightbestemcells equivalenttotheimmortalstemcellincharacterandmortalonlyinthesensethattheirprogenyhappensubsequentlytobejostledoutofthebasallayerandshedfromtheskin Figure22 37Myoblastfusioninculture Thephase contrastmicrographsshowhowthecellswillproliferate lineup andfusetoformmultinucleatemusclecells C isathighermagnification showingthecross striationsthatarejustbeginningtobevisibleasthecontractileapparatusdevelops redarrow andtheaccumulationsofmanynucleiwithinasinglecell greenarrows CourtesyofRosalindZalin Figure22 39Autoradiographofasinglemultinucleatemusclecellwithassociatedsatellitecells ThefiberhasbeenisolatedfromanadultratandtransferredintoculturemediumcontainingH thymidineplusanextractfromdamagedmusclethatstimulatesthesatellitecellstodivide Thedividingsatellitecells arrows havebecomeradioactivelylabeled silvergrainsvisibleasblackdots themusclecellnucleiareunabletoproliferateandremainunlabeled FromR Bischoff Dev Biol 115 140 147 1986 Figure21 99Atypicalneuronofavertebrate Thearrowsindicatethedirectioninwhichsignalsareconveyed Theneuronshownisfromtheretinaofamonkey Thelongestandlargestneuronsinahumanextendforabout1millionmmandhaveanaxondiameterof15mm Figure21 100Thecomplexorganizationofnervecellconnections Thissemischematicdrawingdepictsasectionthroughasmallpartofamammalianbraintheolfactorybulbofadog stainedbytheGolgitechnique Theblackobjectsareneurons thethinlinesareaxonsanddendrites throughwhichthevarioussetsofneuronsareinterconnectedaccordingtopreciserules Figure21 101Thethreephasesofneuraldevelopment Figure21 102Diagramofanearly 21 2 day chickembryo showingtheoriginsofthenervoussystem Theneuraltube lightgreen hasalreadyclosed exceptatthetailend andliesinternally beneaththeectoderm ofwhichitwasoriginallyapart Theneuralcrest red liesdorsallybeneaththeectoderm inorabovetheroofoftheneuraltube Inaddition thickenings orplacodes darkgreen intheectodermoftheheadgiverisetosomeofthesensorytransducercellsandneuronsofthatregion includingthoseoftheearandthenose Thecellsoftheretinaoftheeye bycontrast originateaspartoftheneuraltube Figure21 103Formationoftheneuraltube Thescanningelectronmicrographshowsacross sectionthroughthetrunkofa2 daychickembryo Theneuraltubeisabouttocloseandpinchofffromtheectoderm atthisstageitconsists inthechick ofanepitheliumthatisonlyonecellthick Figure21 109NGFeffectsonneuriteoutgrowth Dark fieldphotomicrographsofasympatheticganglionculturedfor48hourswith above orwithout below NGF NeuritesgrowoutfromthesympatheticneuronsonlyifNGFispresentinthemedium EachculturealsocontainsSchwann glial cellsthathavemigratedoutoftheganglion thesearenotaffectedbyNGF NeuronalsurvivalandmaintenanceofgrowthconesforneuriteextensionrepresenttwodistincteffectsofNGF Theeffectongrowthconesislocal direct rapid andindependentofcommunicationwiththecellbody whenNGFisremoved thedeprivedgrowthconeshalttheirmovementswithinaminuteortwo TheeffectofNGFoncellsurvivalislessimmediateandisassociatedwithuptakeofNGFbyendocytosisanditsintracellulartransportbacktothecellbody 4 7 器官培养Organculture 基本概念part rudiments organizedtissue器官化的组织 organexplant器官型植块 organtypicculture器官型培养发展简史基本原则培养方法 Figure22 43Thegrowthofcartilage Thetissueexpandsasthechondrocytesdivideandmakemorematrix Thefreshlysynthesizedmatrixwithwhicheachcellsurroundsitselfisshadeddarkgreen Cartilagemayalsogrowbyrecruitingfibroblastsfromthesurroundingtissueandconvertingthemintochondrocytes Figure22 44Depositionofbonematrixbyosteoblasts Osteoblastsliningthesurfaceofbonesecretetheorganicmatrixofbone osteoid andareconvertedintoosteocytesastheybecomeembeddedinthismatrix Thematrixcalcifiessoonafterithasbeendeposited Theosteoblaststhemselvesarethoughttoderivefromosteogenicstemcellsthatarecloselyrelatedtofibroblasts Figure22 45Anosteoclastshownincross section Thisgiant multinucleatedcellerodesbonematrix The ruffledborder isasiteofsecretionofacids todissolvetheboneminerals andhydrolases todigesttheorganiccomponentsofthematrix Osteoclastsvaryinshape aremotile andoftensendoutprocessestoresorbboneatmultiplesites Theydevelopfrommonocytesandcanbeviewedasspecializedmacrophages FromR V Krsti c UltrastructureoftheMammalianCell AnAtlas Berlin Springer 1979 Figure22 46Theremodelingofcompactbone Osteoclastsactingtogetherinasmallgroupexcavateatunnelthroughtheoldbone advancingatarateofabout50mmperday Osteoblastsenterthetunnelbehindthem lineitswalls andbegintoformnewbone depositinglayersofmatrixatarateof1or2mmperday Atthesametimeacapillarysproutsdownthecenterofthetunnel Thetunnelwilleventuallybecomefilledwithconcentriclayersofnewbone withonlyanarrowcentralcanalremaining Eachsuchcanal besidesprovidingarouteofaccessforosteoclastsandosteoblasts containsoneormorebloodvesselsbringingthenutrientsthebonecellsmusthavetosurvive Typically about5 10 oftheboneinahealthyadultmammalisreplacedinthiswayeachyear Figure22 47Transversesectionthroughacompactouterportionofalongbone Themicrographshowstheoutlinesoftunnelsformedbyosteoclastsandthenfilledinbyosteoblastsduringsuccessiveroundsofboneremodeling Thesectionhasbeenpreparedbygrinding thehardmatrixhasbeenpreservedbutnotthecells Lacunaeandcanaliculithatwereoccupiedbyosteocytesareclearlyvisible however Thealternatingbrightanddarkconcentricringscorrespondtoanalternatingorientationofthecollagenfibersinthesuccessivelayersofbonematrixlaiddownbytheosteoblaststhatlinedthewallofthecanalduringlife ThispatternisrevealedherebyviewingthespecimenbetweenpartlycrossedPolaroidfilters Notehowoldersystemsofconcentriclayersofbonehavebeenpartlycutthroughandreplacedbynewersystems Figure22 48Thedevelopmentofalongbone Longbones suchasthefemurorthehumerus developfromaminiaturecartilagemodel Uncalcifiedcartilageisshowningreen calcifiedcartilageinblack boneinbrown andbloodvesselsinred Thecartilageisnotconvertedtobonebutisgraduallyreplacedbyitthroughtheactionofosteoclastsandosteoblasts whichinvadethecartilageinassociationwithbloodvessels Osteoclastserodecartilageandbonematrix whileosteoblastssecretebonematrix Theprocessofossificationbeginsintheembryoandisnotcompleteduntiltheendofpuberty Theresultingboneconsistsofathick walledhollowcylinderofcompactboneenclosingalargecentralcavityoccupiedbythebonemarrow Notethatnotallbonesdevelopinthisway Themembranebonesoftheskull forexample areformeddirectlyasbonyplates notfromapriorcartilagemodel Figure22 49Remodelingofalongboneinthelegafterafracturethathashealedoutoftrue Thedeformityintherecentlyhealedboneexposesittoabnormalstresses Wherethecompressiveforcesareincreased therateofbonedepositionisincreasedrelativetotherateoferosion wheretheforcesaredecreased therateofdepositionisdecreasedrelativetotherateoferosion Inthiswaytheboneisgraduallyremodeledbacktoitsnormalform Figure22 13Angiogenesis Anewbloodcapillaryformsbythesproutingofanendothelialcellfromthewallofanexistingsmallvessel Thisschematicdiagramisbasedonobservationsofcellsinthetransparenttailofalivingtadpole AfterC C Speidel Am J Anat 52 1 79 1933 Figure22 14Capillaryformationinvitro Endothelialcellsinculturespontaneouslydevelopinternalvacuolesthatjoinup givingrisetoanetworkofcapillarytubes Photographs A and B showsuccessivestagesintheprocess thearrowin A indicatesavacuoleforminginitiallyinasingleendothelialcell Theculturesaresetupfromsmallpatchesoftwotofourendothelialcellstakenfromshortsegmentsofcapillary Thesecellswillsettleonthesurfaceofacollagen coatedculturedishandformasmallflattenedcolonythatenlargesgraduallyasthecellsproliferate Thecolonyspreadsacrossthedish andeventually afterabout20days capillarytubesbegintoforminthecentralregions Oncetubeformationhasstarted branchessoonappear andafter5to10moredaysanextensivenetworkoftubesisvisible asseenin B Figure22 25Whitebloodcells A D Electronmicrographsshowing respectively aneutrophil abasophil aneosinophil andamonocyte ElectronmicrographsoflymphocytesareshowninFigure23 4 Eachofthecelltypesshownherehasadifferentfunction whichisreflectedinthedistinctivetypesofsecretorygranulesandlysosomesitcontains Thereisonlyonenucleuspercell butithasanirregularlobedshape andin B C and D theconnectionsbetweenthelobesareoutoftheplaneofsection E LightmicrographofabloodsmearstainedwiththeRomanowskystain whichcolorsthewhitebloodcellsstrongly A D courtesyofDorothyBainton E courtesyofDavidMason Figure22 27Bonemarrow A Lightmicrographofastainedsection Thelargeemptyspacescorrespondtofatcells whosefattycontentshavebeendissolvedawayduringspecimenpreparation Thegiantcellwithalobednucleusisamegakaryocyte B Low magnificationelectronmicrograph Thistissueisthemainsourceofnewbloodcells exceptforTlymphocytes whichareproducedinthethymus Notethattheimmaturebloodcellsofaparticulartypetendtoclusterin familygroups A courtesyofDavidMason B fromJ A G Rhodin Histology ATextandAtlas NewYork OxfordUniversityPress 1974 Figure22 30Atentativeschemeofhemopoiesis Thepluripotentstemcellnormallydividesinfrequentlytogenerateeithermorepluripotentstemcells self renewal orcommittedprogenitorcells labeledCFC colony formingcells whichareirreversiblydeterminedtoproduceonlyoneorafewtypesofbloodcells Theprogenitorcellsarestimulatedtoproliferatebyspecificgrowthfactorsbutprogressivelylosetheircapacityfordivisionanddevelopintoterminallydifferentiatedbloodcells whichusuallyliveforonlyafewdaysorweeks InadultmammalsallofthecellsshowndevelopmainlyinthebonemarrowexceptforTlymphocytes whichdevelopinthethymus andmacrophagesandosteoclasts whichdevelopfrombloodmonocytes ThemostcontroversialpartoftheschemeiswheretheprecursorsforTandBlymphocytesfitintothescheme Thedashedlinesreflectthisuncertainty Thepluripotentstemcellsalsogiverisetovarioustypesoftissuecellsnotshowninthisscheme suchasNKcells mastcells andavarietyofclassesofantigen presentingcells discussedinChapter23 butthepathwaysbywhichthesecellsdevelopareuncertain Figure22 32Thedevelopmentofredbloodcells ThedrawingshowstherelationshipbetweentheBFC E theCFC E andthematureerythrocyte BFC EsandCFC Esarebothcommittederythroidprogenitorcells BFC EsrespondtothefactorIL 3butnottoerythropoietin whereasCFC Esrespondtoerythropoietin Theseriesofcelldivisionsthatoccurinthislineageundertheinfluenceoferythropoietinprovidesapowerfulmeansofcontrollingtheproductionoferythrocyteswithoutupsettingtheproductionofothertypesofbloodcells Figure22 16Renewalofthegutlining A Thepatternofcellturnoverandtheproliferationofstemcellsintheepitheliumthatformstheliningofthesmallintestine Thenondividingdifferentiatedcellsatthebaseofthecryptsalsohaveafinitelifetime terminatedbyprogrammedcelldeath andarecontinuallyreplacedbyprogenyofthestemcells B Photographofasectionofpartoftheliningofthesmallintestine showingthevilliandcrypts Notehowmucus secretinggobletcells stainedred areinterspersedamongtheabsorptivebrush bordercellsintheepitheliumofthevilli SeeFigure22 9forthestructureofthesecells 4 8 干细胞 定义特性种类 全 toti 能 多 pluri multi 寡 oligo 和单 uni 能 potent 胚胎干细胞 EC ES EG 成体 组织 干细胞 MilestonesinStemCellResearch MouseEScells EvensMJandKaufman 1981 CloningofDolly Wilmut 1997 EstablishinghumanEScells Thomson 1998 Trans differentiation Goodale Verfaillie 2003 HumanEScellsbySCNT Huang 2005 GenerationofiPScells Yamanaka 2006 8 2ES EG 特性与鉴定获得建系过程体外培养体外诱导分化意义和应用前景 Thedefinitionofastemcell Eachdaughterproducedwhenastemcelldividescaneitherremainastemcellorgoontobecometerminallydifferentiated Figure21 29Theearlystagesofmousede