贵州医科大学学报

2014, v.39;No.171(06) 861-864

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人诱导多能干细胞向血管内皮细胞分化方法的建立
Establishment of the Method for Differentiation of Human Induced Pluripotent Stem Cells into Vascular Endothelial Cells

杨建昌;刘慧;刘莉;
YANG Jianchang;LIU Hui;LIU Li;Department of Cardiology,the First People's Hospital of Guiyang City;Taihe Hospital of Shiyan City;

摘要(Abstract):

目的:建立一种新的促进人诱导多能干细胞(i PSc)向血管内皮细胞(VEC)分化的方法。方法:将未分化的人i PSc制备成拟胚体(EBs),分为常氧组(A组)、常氧加血管内皮生长因子(VEGF)组(B组)、常氧加甲基乙二酰氨基乙酸(DMOG)组(C组)、缺氧组(D组)、缺氧加VEGF组(E组)及缺氧加DMOG组(F组),分别给予相应处理;观察各组i PS-EBs分化的VEC形态结构,应用逆转录PCR(RT-PCR)法检测VEC特异基因CD31、CD34、VE-cad、KDR、v WF的表达,免疫荧光细胞法检测以mRNA表达最强组的VEC特异基因在细胞定位情况判断VEC分化情况。结果:RT-PCR结果显示未经诱导i PSc不表达任何VEC相关基因,A组EBs细胞仅表达微弱的VE-cad和KDR;经条件诱导后i PS-EBs细胞表达CD31、CD34、VE-cad、KDR、v WF,C组表达最强,与其他各组比较差异有统计学意义(P<0.05);免疫荧光结果显示C组中CD31、CD34为膜表达,KDR、v WF为胞浆表达。结论:常氧状态下加入DMOG培养能成功促进人i PSc向VEC分化。
Objective: To establish a new method for differentiation of human induced pluripotent stem cells( i PSc) into vascular endothelial cells( VEC). Methods: Undifferentiated human i PSc were prepared into embryoid bodies( EBs),and then divided into 6 groups as follows: normoxia group( group A),normoxia + vascular endothelial growth factor( VEGF) group( group B),normoxia +DMOG group( group C),hypoxia group( group D),hypoxia + VEGF group( group E),hypoxia +DMOG group( group F). The cells in the 6 groups were given corresponding treatment. VEC morphology in each group was observed,reverse transcription PCR( RT-PCR) and immunofluorescence cell method were employed to detect the expression of VEC specific gene( CD31,CD34,VE-cad,KDR,v WF) expression to determine VEC differentiation condition. Results: RT-PCR showed that i PSc without being induced didn't express any VEC related gene and the cells in group A only weakly expressed VE-cad and KDR; After induced,the cells expressed CD31,CD34,VE-cad,KDR and v WF,and the gene levels of group C were higher than those in the other groups( P < 0. 05). Immunofluorescence cell method showed that CD31,CD34 in group C expressed on the membrane and KDR,v WF expressed in the cytoplasm. Conclusions: Under the conditions of normoxia,DMOG can promote i PSc to differentiate into VEC.

关键词(KeyWords): 甲基乙二酰氨基乙酸;多能干细胞;诱导分化;血管内皮细胞;血管内皮细胞生长因子
DMOG;pluripotent stem cells;induced differentiation;vascular endothelial cells;vascular endothelial growth factor

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作者(Author): 杨建昌;刘慧;刘莉;
YANG Jianchang;LIU Hui;LIU Li;Department of Cardiology,the First People's Hospital of Guiyang City;Taihe Hospital of Shiyan City;

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