小鼠胚胎干细胞分化为血管内皮细胞的永生化研究  

IMMORTALIZATION OF ENDOTHELIAL CELLS DIFFERENTIATED FROM MOUSE EMBRYONIC STEM CELLS

作　　者：沈干[1] 丛笑倩[1] 杜忠伟[2] 吴春芳[1] 刘晓音[1] 刘伟[1] 曹谊林[1] 

机构地区：[1]上海第二医科大学附属第九人民医院整复外科上海市组织工程重点实验室 [2]中国科学院上海生物化学与细胞生物学研究所,上海200031

出　　处：《实验生物学报》2002年第3期218-228,共11页Acta Biologiae Experimentalis Sinica

基　　金：国家重点基础研究发展规划973项目(G1999054300);上海市重点学科"重中之重"项目。

摘　　要：本文探讨了小鼠胚胎干细胞(ES细胞)诱导分化的血管内皮细胞永生化。在体外培养系统中,以维甲酸(RA)和转化生长因子-β1(TGF-β1)诱导小鼠胚胎干细胞(ES细胞)的拟胚体(EB)分化为“圆形细胞”和由这些“圆形细胞”组成的血管样结构。经光学和扫描电镜及免疫荧光等法分析检测,证明组成血管样结构的细胞具有专一性vWF荧光染色,表明是血管内皮样细胞。利用脂质体将人端粒酶催化亚基逆转录酶(hTERT)基因转染诱导分化中的“圆形细胞”。应用Dot-blot,RT-PCR,Western blot及免疫组织化学等方法分析、观察和证明了诱导分化的组成血管样结构的园形细胞和被hTERT基因转染的“圆形”细胞的形态和生物学特性。结果表明,携带hTERT基因的从ES细胞分化来的圆形细胞在体外可大量增殖,持续传代,95%具有血管内皮细胞的一些特有标志和管道化生长特性。因此,通过人端粒酶基因的转染途径可解决由ES细胞诱导分化而来的内皮细胞扩增和永生化问题,为构建组织工程化血管及其它人工血管的内皮化提供种子细胞来源打下基础。This study is designed to immortalize endothelial cells differentiated from embryonic stem cells. The embryoid bodies (EB) formed in vitro from embryonic stem cells, were induced to differentiate into many 'round cells' (the precursor of endothelial cells) by retinoic acid (RA) and transforming growth factor-β1 (TGF-β1). These 'round cells' later formed the vascular tube-like structures. Studies by scanning electronic microscopy and light microscopy and immunocytochemistry, demonstrated that these tube-like structures were constituted by a large number of round and flat cells, which were positive for 'vWF' and 'CD34'staining. These results indicate they are vascular endothelial cells. To immortalize these cells, human telomerase reverse transcriptase ( hTERT) cDNA was transfected into 'round cells' by lipofectine. hTERT mRNA expression in transfected cells was confirmed by Dot blot, RT-PCR. Furthermore ,95% these transfected cells maintain the characteristic of endothelial cell,can proliferate in large quantity in vitro, and are able to form tubular structures. These results suggest that hTERT cDNA transfection can immortalize the induced endothelial cells and therefore may provide a new source of seed cells for vascular engineering.

关 键 词：小鼠 胚胎干细胞分化 血管内皮细胞 永生化 ES细胞 诱导分化 组织工程 人端粒酶催化亚基逆转录酶 

分 类 号：Q25[生物学—细胞生物学]