血小板裂解液對人臍帶間充質(zhì)干細(xì)胞體外成軟骨分化的影響
發(fā)布時(shí)間：2019-07-10 10:05
【摘要】：目的探討血小板裂解液(platelet lysate,PL)在體外定向誘導(dǎo)人臍帶間充質(zhì)干細(xì)胞(human umbilical cord derived mesenchymal stem cells,hUCMSCs)分化成軟骨細(xì)胞中的作用。方法取健康產(chǎn)婦自愿捐贈(zèng)臍帶,采用膠原酶消化法分離hUCMSCs,體外培養(yǎng)擴(kuò)增,流式細(xì)胞儀進(jìn)行細(xì)胞表型鑒定。根據(jù)加入誘導(dǎo)培養(yǎng)基成分不同將實(shí)驗(yàn)分為以下3組:A組為H-DMEM培養(yǎng)基、10%FBS及10%PL,B組為H-DMEM培養(yǎng)基、10%FBS、10 ng/mL TGF-β1、1×10-7 mol/L地塞米松、50μg/mL維生素C及1%胰島素鐵硒傳遞蛋白(insulin-transferrin-selenium,ITS),C組為H-DMEM培養(yǎng)基、10%FBS、10 ng/mL TGF-β1、1×10-7 mol/L地塞米松、50μg/mL維生素C、1%ITS及10%PL。誘導(dǎo)培養(yǎng)2周,甲苯胺藍(lán)染色檢測各組軟骨細(xì)胞基質(zhì)的分泌,免疫熒光檢測軟骨特異性Ⅱ型膠原表達(dá),半定量RT-PCR檢測蛋白聚糖(Aggrecan)和Ⅱ型膠原表達(dá)。結(jié)果分離得到的hUCMSCs不表達(dá)造血細(xì)胞的表面標(biāo)記CD45、CD34和HLA-DR,而表達(dá)黏附分子和MSCs表面標(biāo)記CD44、CD105和CD146。甲苯胺藍(lán)染色和Ⅱ型膠原免疫熒光染色示C組呈陽性,B組呈弱陽性,而A組均呈陰性。半定量RT-PCR檢測示Aggrecan和Ⅱ型膠原在B、C組中均有表達(dá),A組中未見表達(dá);C組Aggrecan mRNA和Ⅱ型膠原mRNA表達(dá)明顯高于B組,差異均有統(tǒng)計(jì)學(xué)意義(P0.05)。結(jié)論單純10%PL不能誘導(dǎo)hUCMSCs成軟骨分化,但它可當(dāng)作成軟骨誘導(dǎo)培養(yǎng)基的輔助添加劑,對hUCMSCs成軟骨分化有明顯促進(jìn)作用,為構(gòu)建組織工程軟骨提供了新的可利用條件。
文內(nèi)圖片：
圖片說明：hUCMSCs 形態(tài)學(xué)觀察（倒置相差顯微鏡 × 100）
[Abstract]:Objective To study the role of platelet lysate (PL) in the differentiation of human umbilical cord mesenchymal stem cells (hUCMSCs) into chondrocytes in vitro. Methods The umbilical cord of healthy pregnant women was donated voluntarily, hUCMSCs were isolated by collagenase digestion, and the cell phenotype was identified by flow cytometry. The experiment was divided into the following three groups according to the different composition of the induction medium: the A group was H-DMEM medium,10% FBS and 10% PL, and the B group was H-DMEM medium,10% FBS,10 ng/ mL TGF-1,1-10-7 mol/ L dexamethasone,50. m u.g/ mL of vitamin C and 1% insulin-iron-selenium-transmitting protein (ITS). Group C was H-DMEM medium,10% FBS,10 ng/ mL TGF-1,1%10-7 mol/ L dexamethasone,50. m u.g/ mL of vitamin C,1% ITS and 10% PL. The expression of cartilage-specific type-II collagen, semi-quantitative RT-PCR (Agomelan) and type II collagen was detected by immunofluorescence,2-week induction culture and toluidine blue staining. Results The surface markers CD45, CD34 and HLA-DR of the hematopoietic cells were not expressed by the isolated hUCMSCs, and the expression of CD44, CD105 and CD146 on the surface of the adhesion molecule and the MSCs. The staining of toluidine blue and the immunofluorescence staining of type 鈪，
本文編號：2512533
文內(nèi)圖片：
圖片說明：hUCMSCs 形態(tài)學(xué)觀察（倒置相差顯微鏡 × 100）
[Abstract]:Objective To study the role of platelet lysate (PL) in the differentiation of human umbilical cord mesenchymal stem cells (hUCMSCs) into chondrocytes in vitro. Methods The umbilical cord of healthy pregnant women was donated voluntarily, hUCMSCs were isolated by collagenase digestion, and the cell phenotype was identified by flow cytometry. The experiment was divided into the following three groups according to the different composition of the induction medium: the A group was H-DMEM medium,10% FBS and 10% PL, and the B group was H-DMEM medium,10% FBS,10 ng/ mL TGF-1,1-10-7 mol/ L dexamethasone,50. m u.g/ mL of vitamin C and 1% insulin-iron-selenium-transmitting protein (ITS). Group C was H-DMEM medium,10% FBS,10 ng/ mL TGF-1,1%10-7 mol/ L dexamethasone,50. m u.g/ mL of vitamin C,1% ITS and 10% PL. The expression of cartilage-specific type-II collagen, semi-quantitative RT-PCR (Agomelan) and type II collagen was detected by immunofluorescence,2-week induction culture and toluidine blue staining. Results The surface markers CD45, CD34 and HLA-DR of the hematopoietic cells were not expressed by the isolated hUCMSCs, and the expression of CD44, CD105 and CD146 on the surface of the adhesion molecule and the MSCs. The staining of toluidine blue and the immunofluorescence staining of type 鈪，
本文編號：2512533
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