人類間質幹細胞之體外增生與分化探討

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結果發現,骨髓間質幹細胞在相同的接種密度下, Mesenchymal stem cells (MSC) can ... 能力與多能性分化潛能的貼附型細胞,近年來因其再生醫學之應用潛力而廣受注目。

Skipnavigation YouareHere:NationalChiaoTungUniversityInstitutionalRepository Publications Thesis Title: 人類間質幹細胞之體外增生與分化探討TheInVitroProliferationandDifferentiationofHumanMesenchymalStemCells Authors: 孫立易Sun,Li-Yi邱顯泰邱紫文Chiu,Hsien-TaiChiou,Tzyy-Wen生物科技學系 Keywords: 間質幹細胞;脈衝式電磁場;生長動力學;微載體;分化潛能;骨生成;Mesenchymalstemcells;Pulsedelectromagneticfields;Growthkinetics;Microcarriers;Differentiationpotential;Osteogenesis IssueDate: 2010 Abstract: 間質幹細胞(MSC)廣泛存在於人體各處,是ㄧ種具有高度體外增生能力與多能性分化潛能的貼附型細胞,近年來因其再生醫學之應用潛力而廣受注目。

本研究首先探討,骨髓間質幹細胞(BMMSC)在脈衝電磁場(PEMF)臨床應用於加速骨癒合之角色,以不同骨分化位階之骨髓間質幹細胞來研究相同條件之脈衝式電磁場對於細胞之刺激現象。

實驗共分為三組,分別為BMMSC組(未分化骨髓間質幹細胞)、Osteogenic組(骨髓間質幹細胞照射時同步進行骨分化誘導)以及Osteoblast組(已進行骨誘導7日之骨髓間質幹細胞)。

三組實驗組皆每天照射脈衝式電磁場8小時,且另設不照射脈衝式電磁場之對照組。

結果發現,每組的實驗組皆會在第一天出現細胞密度顯著較高的現象,且細胞週期分析與重複實驗也佐證了此現象確實存在。

但是,BMMSC組與Osteogenic組的最高可達細胞密度會相差九倍;Osteoblast組在同樣的脈衝式電磁場作用下,只有第一天實驗組細胞密度較高之後反而會相反。

此外,脈衝式電磁場照射會加速Osteogenic組骨分化前期(1~7天)之鹼性磷酸酶細胞提早出現且多,但直到骨分化中期(11天)才會發生鈣堆積量顯著增加的現象。

從基因變化也可以發現,脈衝式電磁場會顯著加速Osteogenic組之骨相關基因提早表現,特別是cbfa1。

故因此推論,脈衝式電磁場對於不同分化位階之骨髓間質幹細胞,應該會造成不同的影響。

當骨髓間質幹細胞維持幹細胞狀態時,脈衝式電磁場可提高其細胞增生現象而不顯著改變其特性。

但是,當骨髓間質幹細胞開始進行骨分化後,脈衝式電磁場除提升其細胞增生現象外,亦會加速其骨分化進程與增強骨形成作用。

最後,當骨髓間質幹細胞已分化為較成熟之成骨細胞後,脈衝式電磁場只會短暫提升其細胞增生現象,卻很快會對其增生發生抑制。

這樣的發現,應可部份解釋之前各研究成果彼此矛盾的現象,讓我們更進一步了解脈衝式電磁場的作用機制。

另外,為探討人類間質幹細胞利用生物可分解之微載體懸浮培養系統之可行性及其特性變化,本研究藉由探討半連續式平面培養(PC組)、半連續式微載體培養(MC組)及饋料式微載體培養(MCBead-T組)這三種培養策略對骨髓間質幹細胞及脂肪間質幹細胞的生長動力學、細胞外型、族群、表現型及分化能力等特性影響,來了解微載體培養間質幹細胞之最佳化程序設計。

結果發現,骨髓間質幹細胞在相同的接種密度下,平面培養與微載體培養之骨髓間質幹細胞的細胞增生倍數接近,但是不同的培養系統會影響其生長延遲期。

此外,MC組培養骨髓間質幹細胞過程中似具挑選作用,產出的細胞顆粒性較低且較能保持趨化因子受體CXCR4表現強度,而且這些MC組培養過3天後之骨髓間質幹細胞,會在骨分化以及脂肪分化上具有較強的潛能。

以MCBead-T組培養骨髓間質幹細胞雖可因此延長細胞培養時間,且數倍提高了此培養系統之可得細胞數,但此細胞會因此發生細胞大小及顆粒性變化,且骨分化及脂肪分化上表現也較差。

然而,之前出現在骨髓間質幹細胞的細胞均質化現象並不會顯著發生在脂肪間質幹細胞,且MC組培養3天後之脂肪間質幹細胞不會如骨髓間質幹細胞般保持其CXCR4表現強度。

雖然,本研究成功將脂肪間質幹細胞培養於無血清之微載體系統中,不但具更快之生長速率且仍保持其分化潛能,但此系統培養之脂肪間質幹細胞在骨分化以及脂肪分化潛能變化並不同於骨髓間質幹細胞。

故本研究可得以下結論,間質幹細胞確實可利用攪拌式微載體培養系統進行體外擴增,並擁有進一步規模化之可能,但當採用不同培養策略或不同種類之間質幹細胞時,所得之細胞品質會具有差異性。

Mesenchymalstemcells(MSC)canbeisolatedfromalmostanytissueofthebodyhavebeenrecognizedtoconstituteapowerfultoolinregenerativemedicineduetotheirmulti-lineagedifferentiationabilityandtheircapacityfortissuerepair. Pulsedelectromagneticfields(PEMF)havebeenclinicallyemployedformanyyears.Despitetheclinicalsuccess,therearecontradictorydataconcerningtheeffectofPEMFstimulationoninvitroproliferationofsomeosteogeniccelllinesorprimaryosteoblasts,soitisafactthatthemechanismunderlyinghowPEMFpromotestheformationofboneoncellularlevelisstillnotfullyunderstood.Forthefirsttime,theeffectofPEMFexposuretostemcellswasdescribedinthisstudy.WediscoveredthatashorterlagphaseandahigherpercentageofG0/G1phaseofbonemarrowmesenchymalstemcells(BMMSC)afterthefirstPEMFtreatment.Althoughthesurfacephenotype,morphology,differentiationpotentialandgrowthratesofBMMSCduringtheexponentialgrowthphasewerenotsignificantlyaffected,thecelldensitiesachievedbyPEMFstimulationweresignificantlyhigherthanthoseachievedinnon-treatedconditions.ThisobservationofacceleratedgrowthofBMMSCduetoPEMFprovidesapossibleexplanationfortheclinicalsuccess.Inaddition,accordingtoabove-mentionedreportsandournewdata,ourhypothesiswasthatPEMFnotonlymodifiestheosteogenesisofBMMSCbutalsoinducesdifferentresponseofcellproliferationdependingontheosteogenicstageofcells.ThisfindinghelpsustounderstandmoreabouttheinvitroandinvivointeractionofPEMFswithbonecells.Furthermore,sincethemorphologyandthemulti-lineagedifferentiationpotentialoftheBMMSCwerenotsignificantlychangedbythePEMF,thestirredbioreactorcancombinewithmicrocarriersandPEMFdevicetobeapowerfultoolforinvitroBMMSCexpansioninthefuture. Duetoitsprovisionofhighspecificsurfaceareaandthree-dimensionalculturecondition,microcarrierculture(MC)hasgarneredgreatinterestforitspotentialtoexpandanchorage-dependentstemcells.Thisstudyutilisessemi-continuousMCascomparedwithcontrolplateculture(PC)orserialbead-to-beadtransferMC(MCBead-T)forinvitroexpansionofhumanMSCincludingofBMMSCandadipose-drivedstemcell(ADSC),andanalysesitseffectsongrowthkinetics,cellphenotypes,andthedifferentiationpotential.ThemaximumcelldensityandoverallfoldincreaseincellgrowthweresimilarbetweenPCsandMCswithsimilarstartingconditions,butthelagphaseofBMMSCgrowthdifferedsubstantiallybetweenthetwogrowthconditions;moreover,MCcellsexhibitedreducedgranularityandhigherCXCR4expression.DifferentiationofBMMSCintoosteogenicandadipogeniclineageswasenhancedafter3daysinMC.However,MCBead-Tresultedinchangesincellgranularityandlowerosteogenicandadipogenicdifferentiationpotential.However,theresultsofMCcellsexhibitedreducedgranularityandhigherC-X-Cchemokinereceptortype4(CXCR-4)expressionswerenotexistinADSC.AlthoughADSCcouldproliferateinMCwithserum-freemediumwithhighergrowthratethanBMMSCorADSCinMC,theosteogenicandadipogeniclineageswerenotenhancedafter3daysinMC.Inconclusion,MCcouldsupporttheexpansionofMSCinascalablethree-dimensionalculturesystem,butthedifferenttypesofMSCordifferentculturesystemswouldresultdifferentialqualityofstemcellhomingabilityandosteogenicandadipogenicdifferentiationofMSC. URI: http://140.113.39.130/cdrfb3/record/nctu/#GT079328808http://hdl.handle.net/11536/40608 AppearsinCollections:Thesis IR@NYCUCrossRefPulsedElectromagneticFieldsAccelerateProliferationandOsteogenicGeneExpressioninHumanBoneMarrowMesenchymalStemCellsDuringOsteogenicDifferentiation/Sun,Li-Yi;Hsieh,Dean-Kuo;Lin,Po-Cheng;Chiu,Hsien-Tai;Chiou,Tzyy-WenCellproliferationofhumanbonemarrowmesenchymalstemcellsonbiodegradablemicrocarriersenhancesinvitrodifferentiationpotential/Sun,L.-Y.;Hsieh,D.-K.;Syu,W.-S.;Li,Y.-S.;Chiu,H.-T.FunctionalCellsCulturedonMicrocarriersforUseinRegenerativeMedicineResearch/Sun,Li-Yi;Lin,Shinn-Zong;Li,Yuan-Sheng;Harn,Horng-Jyh;Chiou,Tzyy-WenTheEffectsofDifferentDynamicCultureSystemsonCellProliferationandOsteogenicDifferentiationinHumanMesenchymalStemCells/Tsai,Hsiou-Hsin;Yang,Kai-Chiang;Wu,Meng-Huang;Chen,Jung-Chih;Tseng,Ching-LiLoading...



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