【摘要】 目的:观察胚胎干细胞在转化生长因子β1和血管内皮细胞生长因子体外诱导分化血管内皮细胞的能力,探讨提高诱导效率的方法。方法:实验于2004/2006在南昌大学医学院第二附属医院血液病研究所进行。实验材料:成年昆明小鼠由南昌大学医学院动物科学部提供(机构许可证号:96021),经自然交配怀孕。小鼠胚胎干细胞129×1/SvJ细胞系,购于ATCC公司。实验方法:取孕12.5～14.5d的胎鼠,制作小鼠胚胎成纤维细胞饲养层。在小鼠胚胎成纤维细胞饲养层上培养扩增胚胎干细胞。将饲养层上扩增后生长状态好的胚胎干细胞以0.25%胰酶消化,小心吹打成单个细胞悬液,以差速贴壁法将饲养层细胞去掉,按1×104L-1胚胎干细胞悬液悬滴在细胞培养皿的盖上,每滴10～20μL,不换液,3d后形成拟胚体,培养液为不含白血病抑制因子的胚胎干细胞培养液。挑选状态好的拟胚体分3组:转化生长因子β1诱导组、血管内皮细胞生长因子诱导组、转化生长因子β1+血管内皮细胞生长因子诱导组。采用RT-PCR和免疫组织化学方法证实诱导的细胞是否为内皮细胞。结果:①胚胎干细胞体外诱导生长观察:3组中拟胚体贴壁后第2天,胚体均略摊开,周围有上皮样细胞出现,第3,4天有许多卵石样细胞产生,至第6天开始出现由卵石样细胞构成的管状结构,自胚体向周围呈辐射状生长,渐呈网状,诱导时间约2周。转化生长因子β1+血管内皮细胞生长因子诱导组中产生的管样结构的拟胚体数较转化生长因子β1诱导组、血管内皮细胞生长因子诱导组多(44.67±3.88,28.17±6.08,33.00±2.68,P<0.05)。②RT-PCR法检测基因mRNA水平:3组基因表达的扩增片断分别为1086,733,265bp,与DNAmark相比较,条带位置相符。③扩增细胞的免疫组织化学染色:Ⅷ因子抗体反应显示阳性。结论:转化生长因子β1与血管内皮细胞生长因子均能诱导胚胎干细胞为内皮细胞,两者合用有可能提高诱导效率。更多还原

【Abstract】 AIM: To observe the competence of embryonic stem cells (ESCs) differentiating into vascular endothelial cells in vitro induced by transforming growth factor β1 (TGFβ1) and vascular endothelial cell growth factor (VEGF) and explore the method of elevating inducible efficiency. METHODS: The experiment was carried out in Hematopathy Institute, Second Affiliated Hospital, Medical College of Nanchang University between 2004 and 2006. Experimental materials: Adult Kunming mice provided by Animal Science Department of Medical College of Nanchang University (agency license number 96021) were pregnant by wild pairing. The 129×1/SvJ embryonic stem cell strain was bought from American Type Culture Collection Company. Experimental methods: Foetal mice aged 12.5-14.5 days were selected to manufacture mouse embryonic fibroblast feeder layer. ESCs were amplified on the fibroblast feeder layer. The ESCs under fine growth condition were 0.25% trypsinized, and then briefly blown to become single cell suspension. The fibroblasts were abscised by differential adhesion method. Cells were cultivated in hanging drops (1×104 L-1) on the cover of cell culture dishes, 10-20 μL each drop and cultured without further feeding. After 3 days, cells turned into embryoid bodies. The ESCs culture solution was without leukaemia inhibitory factor. The fine embryoid bodies were collected and divided into 3 groups: TGFβ1 group, VEGF group, TGFβ1 plus VEGF group. Endothelial cells were confirmed by RT-PCR and immunohistochemical method. RESULTS: ①in vitro observation of ESCs growth: At day 2, embryoid bodies of the 3 groups were all briefly spread and epithelioid cell appeared around them. Pebble appearance cells could be seen around them at days 3 and 4. The pebble appearance cells formed tubiform structure that grew radiat from the embryoid bodies at day 6. Induction was for 2 weeks approximately. The number of embryoid bodies with tubiform structure in the TGFβ1 plus VEGF group was more than that in the TGFβ1 group and VEGF group (44.67±3.88,28.17±6.08,33.00±2.68, P < 0.05). ②mRNA level detected by RT-PCR: The three gene fragments were 1 086, 733, 265 bp, respectively. Compared with the strap of DNAmark, position of the three kinds of gene expression amplification fragments were matched. ③immunohistochemical staining in amplified cells: Ⅷ factor antibody response was positive.CONCLUSION: TGFβ1 and VEGF can induce ESCs into endothelial cells and their combination maybe raise the induction efficiency.更多还原