【摘要】： 目的探討人臍帶間充質(zhì)干細(xì)胞分離、培養(yǎng)的方法,研究其基本生物學(xué)特性。初步探討人臍帶間充質(zhì)干細(xì)胞向肝樣細(xì)胞誘導(dǎo)分化的方法。 方法無菌條件下收集剖宮產(chǎn)新生兒臍帶,利用PBS沖洗臍動(dòng)脈及臍靜脈,將血液沖洗盡,將其剪碎至1mm~3后,加入Ⅳ型膠原酶(0.1%)消化60min,然后加入胰酶(0.125%)消化30min,利用濾網(wǎng)過濾后收集細(xì)胞,DMEM(5%FBS)重懸細(xì)胞接種于培養(yǎng)瓶?jī)?nèi)進(jìn)行原代培養(yǎng),待細(xì)胞生長(zhǎng)至80-90%融合后進(jìn)行1:3傳代培養(yǎng),取傳代培養(yǎng)細(xì)胞進(jìn)行形態(tài)學(xué)觀察、免疫細(xì)胞化學(xué)染色、流式細(xì)胞儀檢測(cè)、生長(zhǎng)活性、細(xì)胞周期分析、染色體數(shù)目分析及軟瓊脂克隆形成實(shí)驗(yàn)。進(jìn)一步取第6代細(xì)胞經(jīng)胰酶消化后接種于12孔板中,待細(xì)胞生長(zhǎng)至約70%融合后,利用兩步誘導(dǎo)法進(jìn)行誘導(dǎo),對(duì)照組用原培養(yǎng)液培養(yǎng)。誘導(dǎo)組中加入0.5μM/L地塞米松,50 g/L ITS,20μg/L HGF,10μg/L FGF4,煙堿0.61g/L,共10天。后期加入20μg/L OSM,0.5μM/L地塞米松,50 g/LITS。每3d換液1次,在不同誘導(dǎo)階段收集細(xì)胞后觀察細(xì)胞形態(tài),免疫細(xì)胞化學(xué),RT-PCR,糖原染色。 結(jié)果利用膠原酶及胰酶兩步消化法能充分消化臍帶,細(xì)胞易于獲得,收集細(xì)胞后進(jìn)行原代培養(yǎng),24小時(shí)可觀察到貼壁細(xì)胞,待生長(zhǎng)至10天時(shí)可見細(xì)胞長(zhǎng)滿瓶底,進(jìn)行傳代培養(yǎng),每周傳代1次。觀察細(xì)胞形態(tài)為長(zhǎng)梭形,不規(guī)則形,細(xì)胞大小不一,類似成纖維細(xì)胞,呈漩渦樣生長(zhǎng)。取第6-9代細(xì)胞行免疫細(xì)胞化學(xué)染色結(jié)果顯示細(xì)胞強(qiáng)表達(dá)CD105、Vimentin,基本不表達(dá)CD34、CD31、CD133。經(jīng)流式細(xì)胞儀檢測(cè)表達(dá)CD29、CD49、CD105,基本不表達(dá)造血細(xì)胞標(biāo)志CD34。MTT實(shí)驗(yàn)顯示細(xì)胞倍增時(shí)間為22h。細(xì)胞周期分析表明75%-85%細(xì)胞處于G_0/G_1。染色體分析可見細(xì)胞染色體條數(shù)正常。在軟瓊脂中無克隆形成。經(jīng)誘導(dǎo)后的細(xì)胞在培養(yǎng)到第14d可見細(xì)胞形態(tài)出現(xiàn)變化,細(xì)胞長(zhǎng)突起逐漸消失,逐漸轉(zhuǎn)為圓形或卵圓形,經(jīng)免疫組化檢測(cè)可見細(xì)胞在第1周開始表達(dá)AFP,第2周時(shí)CK18、CK19、OV6的表達(dá)被觀察到。在誘導(dǎo)第3周時(shí)ALB表達(dá)被觀察到,AFP表達(dá)逐漸減弱。到第4周AFP的表達(dá)基本消失,ALB的表達(dá)增強(qiáng)。RT-PCR結(jié)果顯示細(xì)胞于第1周開始表達(dá)AFP,至誘導(dǎo)后期,AFP表達(dá)水平逐漸降低。于第3周時(shí)觀察到ALB表達(dá),隨時(shí)間延長(zhǎng)ALB表達(dá)逐漸增強(qiáng)。于第4周糖原染色呈陽性結(jié)果。 結(jié)論:經(jīng)酶消化法可從臍帶中獲得間充質(zhì)干細(xì)胞,易于獲得,體外增殖能力強(qiáng),與骨髓來源間充質(zhì)干細(xì)胞的免疫表型相一致,符合間充質(zhì)干細(xì)胞基本的生物學(xué)特性,有望在干細(xì)胞的研究及臨床應(yīng)用方面發(fā)揮重要的作用。臍帶間充質(zhì)干細(xì)胞在本實(shí)驗(yàn)誘導(dǎo)條件下可轉(zhuǎn)化為肝樣細(xì)胞,表達(dá)肝細(xì)胞的表面標(biāo)志,初步具備肝細(xì)胞特有的功能性特征,將為肝細(xì)胞組織工程提供理想的細(xì)胞來源。
[Abstract]:Objective To study the isolation and culture of human umbilical cord mesenchymal stem cells, and to study its basic biological characteristics. The method of inducing differentiation of human umbilical cord mesenchymal stem cells to liver-like cells was discussed. Methods The umbilical cord of the newborn was collected under aseptic condition. The umbilical artery and the umbilical vein were washed with PBS. The blood was washed with PBS. The umbilical vein and the umbilical vein were washed. After the blood was cut to 1mm ~ 3, type 鈪，

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