人臍帶間充質(zhì)干細(xì)胞的分離培養(yǎng)及向成骨成脂分化的實驗研究

《人臍帶間充質(zhì)干細(xì)胞的分離培養(yǎng)及向成骨成脂分化的實驗研究》由會員上傳分享，免費在線閱讀，更多相關(guān)內(nèi)容在學(xué)術(shù)論文-天天文庫。

1、人臍帶間充質(zhì)干細(xì)胞的分離培養(yǎng)及向成骨成脂分化的實驗研究作者：孫國棟，李志忠，王晶，林永新，洪亮，吳波文，焦根龍，邵建立【摘要】　　目的探討并優(yōu)化人臍帶間充質(zhì)干細(xì)胞(hUCMSCs)體外獲取及培養(yǎng)增殖的方法并鑒定；誘導(dǎo)其向成骨細(xì)胞和脂肪細(xì)胞分化，為其進(jìn)一步應(yīng)用奠定基礎(chǔ)。方法用酶消化法分離培養(yǎng)hUCMSCs，通過傳代培養(yǎng)、擴(kuò)增，倒置光學(xué)顯微鏡及原子力顯微鏡下觀察細(xì)胞形態(tài)；MTT法檢測細(xì)胞增殖曲線，流式細(xì)胞儀檢測免疫表型；體外向成骨、脂肪方向誘導(dǎo)分化，并通過特異性染色鑒定其分化能力。結(jié)果采用酶消化法能有效分離純化hUCMSCs。接種24h，貼

2、壁細(xì)胞形態(tài)多為長梭型、多邊形或成纖維細(xì)胞樣形態(tài)，大小均一。P3、P7代細(xì)胞的生長曲線基本一致，增殖能力強(qiáng)。流式細(xì)胞儀分析，第3代細(xì)胞強(qiáng)烈表達(dá)CD29、CD44、CD105，不表達(dá)CD34、CD45和HLADR。經(jīng)成骨誘導(dǎo)分化后4周，堿性磷酸酶染色呈強(qiáng)陽性，茜素紅染色可見明顯鈣結(jié)節(jié)；經(jīng)成脂誘導(dǎo)3周，油紅O染色呈陽性，有明顯的脂滴出現(xiàn)。結(jié)論本實驗建立了一套體外穩(wěn)定培養(yǎng)擴(kuò)增hUCMSCs的方法。所培養(yǎng)的細(xì)胞成分單一、擴(kuò)增迅速、生物學(xué)形狀穩(wěn)定，并能使其在體外誘導(dǎo)向成骨細(xì)胞和脂肪細(xì)胞分化，有望成為組織工程較為理想的種子細(xì)胞?！娟P(guān)鍵詞】臍帶；間充

3、質(zhì)干細(xì)胞；分化；成骨細(xì)胞；脂肪細(xì)胞14　　ABSTRACT:ObjectiveToestablishanoptimizedmethodtoisolate,cultureandidentifyhumanumbilicalcordmesenchymalstemcells(hUCMSCs)invitroandinducetheirosteogenicandadipogenicdifferentiation.MethodsThehUCMSCswereisolatedfromhumanumbilicalcordbydigestionwithcol

4、lagenase.Afterserialsubcultivationinvitro,thestemcellswerepassaged.MorphologicappearanceofhUCMSCswasobservedunderanopticalmicroscopeandatomicforcemicroscope.TheproliferationratewasmeasuredbyMTTassay.Cellcycleandsurfaceantigensweremeasuredbyflowcytometry.Theosteogenicanda

5、dipogenicdifferentiationwastestedandevaluatedbyspecificstainingmethods.ResultsTheisolationofhUCMSCsbydigestionwithcollagenasewasefficient.Afterseededfor24hours,theadherentcellsshowedspindleshapeandfibroblastcelllikeshapeandthesizeofhUCMSCswashomogeneous.Thesimilargrowth

6、curvesofpassage3and7exhibitedagreatpotentialforproliferation.FlowcytometryanalysisrevealedthatCD29,CD44andCD105werehighlyexpressedonthesurfaceofpassages3cells,buttheexpressionwasnegativeforCD34,CD45andHLADR.Aftercultureininducingmedium,thecellsweresuccessfullyinducedint

7、oosteogenicandadipogeniclineages.Thesecells14werehighlypositiveforalkalinephosphatestainingandalsoshowedmineralizationpresentedwithvonkossastainingafter4weekscultureinductionofosteogenicdifferentiation.Furthermore,liquidvacuolesweredetectedbyoilredOstainingafter3weeksc

8、ultureinductionofadipogenicdifferentiation.ConclusionAninvitromethodforisolationandpurificationofhUCMSC