中华整形外科杂志
中華整形外科雜誌
중화정형외과잡지
CHINESE JOURNAL OF PLASTIC SURGERY
2014年
3期
191-196
,共6页
傅歆%刘文博%谢方南%肖苒
傅歆%劉文博%謝方南%肖苒
부흠%류문박%사방남%초염
人胚胎干细胞%视黄酸%拟胚体%胚胎发育
人胚胎榦細胞%視黃痠%擬胚體%胚胎髮育
인배태간세포%시황산%의배체%배태발육
Human embryonic stem cells%Retinoic acid%Embryoid body%Embryonic
目的 研究视黄酸对人胚胎干细胞(ESCs)的增殖、干性及其向拟胚体分化的能力的影响,探索应用人ESC为模型研究先天发育畸形机制的可行性.方法 用real-time PCR、MTS细胞增殖分析和免疫荧光染色,鉴定视黄酸处理前、后H9 ESC增殖和干性的变化;用real-time PCR比较H9 ESC向拟胚体分化后,视黄酸处理对三胚层标志基因及成骨和成脂相关基因表达的影响.结果 随着视黄酸处理时间和浓度的增加,H9 ESC的增殖速度在指数生长早期显著上调,干性标志基因OCT4,Nanog和Sox2及Oct4翻译调控因子Lin28的mRNA表达水平显著下降,且Oct4蛋白表达水平也明显降低.视黄酸处理后拟胚体出现空泡结构,外胚层标志基因NeuroD1、Noggin,中胚层标志基因Brachyury、Twist和内胚层标志基因AFP,GATA-4的表达显著上调(P＜0.05),并以AFP的变化最为显著(P＜0.01).此外,成脂相关基因C/EB Pα表达水平也显著提高,但成骨相关基因OPN的表达水平在视黄酸处理5d后显著下调(P＜0.05).结论 高浓度的视黄酸可诱导H9 ESC丧失干性,并使其向拟胚体方向发生过度分化,同时破坏早期拟胚体形成过程中成骨成脂分化的平衡,这可能与其诱发先天性颅面发育畸形相关.
目的 研究視黃痠對人胚胎榦細胞(ESCs)的增殖、榦性及其嚮擬胚體分化的能力的影響,探索應用人ESC為模型研究先天髮育畸形機製的可行性.方法 用real-time PCR、MTS細胞增殖分析和免疫熒光染色,鑒定視黃痠處理前、後H9 ESC增殖和榦性的變化;用real-time PCR比較H9 ESC嚮擬胚體分化後,視黃痠處理對三胚層標誌基因及成骨和成脂相關基因錶達的影響.結果 隨著視黃痠處理時間和濃度的增加,H9 ESC的增殖速度在指數生長早期顯著上調,榦性標誌基因OCT4,Nanog和Sox2及Oct4翻譯調控因子Lin28的mRNA錶達水平顯著下降,且Oct4蛋白錶達水平也明顯降低.視黃痠處理後擬胚體齣現空泡結構,外胚層標誌基因NeuroD1、Noggin,中胚層標誌基因Brachyury、Twist和內胚層標誌基因AFP,GATA-4的錶達顯著上調(P＜0.05),併以AFP的變化最為顯著(P＜0.01).此外,成脂相關基因C/EB Pα錶達水平也顯著提高,但成骨相關基因OPN的錶達水平在視黃痠處理5d後顯著下調(P＜0.05).結論 高濃度的視黃痠可誘導H9 ESC喪失榦性,併使其嚮擬胚體方嚮髮生過度分化,同時破壞早期擬胚體形成過程中成骨成脂分化的平衡,這可能與其誘髮先天性顱麵髮育畸形相關.
목적 연구시황산대인배태간세포(ESCs)적증식、간성급기향의배체분화적능력적영향,탐색응용인ESC위모형연구선천발육기형궤제적가행성.방법 용real-time PCR、MTS세포증식분석화면역형광염색,감정시황산처리전、후H9 ESC증식화간성적변화;용real-time PCR비교H9 ESC향의배체분화후,시황산처리대삼배층표지기인급성골화성지상관기인표체적영향.결과 수착시황산처리시간화농도적증가,H9 ESC적증식속도재지수생장조기현저상조,간성표지기인OCT4,Nanog화Sox2급Oct4번역조공인자Lin28적mRNA표체수평현저하강,차Oct4단백표체수평야명현강저.시황산처리후의배체출현공포결구,외배층표지기인NeuroD1、Noggin,중배층표지기인Brachyury、Twist화내배층표지기인AFP,GATA-4적표체현저상조(P＜0.05),병이AFP적변화최위현저(P＜0.01).차외,성지상관기인C/EB Pα표체수평야현저제고,단성골상관기인OPN적표체수평재시황산처리5d후현저하조(P＜0.05).결론 고농도적시황산가유도H9 ESC상실간성,병사기향의배체방향발생과도분화,동시파배조기의배체형성과정중성골성지분화적평형,저가능여기유발선천성로면발육기형상관.
Objective To analyze the influence of retinoic acid (RA) on the undifferentiated state and EB formation abilities of human embryonic stem cells.Methods The biological characteristics of H9 ESCs after RA treatment were characterized by real-time PCR,MTS proliferation assay and immunofluorescence staining.The expression of three germ layers markers,osteogenic differentiation markers and adipogenic differentiation markers in H9-differentiated embryoid bodies (EBs) with RA treatment were quantified by real time PCR.Results The proliferation of H9 ESCs in the early logarithmic growth phase was accelerated by RA treatment.In addition,RA induced differentiation of H9 ESC coupled with morphology changes,decreased expression of undifferentiated markers Oct4,Nanog,Sox2 and OCT4 mRNA binding protein Lin28 at mRNA level,and reduced expression of Oct4 at protein level.RA induced formation of cavities in EBs.Real time PCR results showed that the expressions of ectodermal markers:NeuroD1,Noggin; mesodermal markers:Brachyury,Twist and endodermal markers:AFP,GATA-4 were significantly increased (P ＜ 0.05),especially for AFP (P ＜ 0.01),by RA treatment in a dose-dependent manner.In addition,the expression of adipogenic differentiation marker C/EBPα was increased while the osteogenic differentiation marker OPN was decreased in EBs after RA treatment for 5 days.Conclusions High concentrations of RA induced the loss of stemness in H9 ESCs and excessive differentiation in EBs,and damaged the balance between osteogenic and adipogenic differentiation during early EB differentiation,which may be relevant to the congenital malformations.
