王志华,何志旭,米蔷,汪浩文
WANG Zhihua,HE Zhixu,MI Qiang,WANG Haowen(Department of Pediatrics

• 1:贵阳医学院干细胞研究中心
• 2:贵阳医学院附院儿科

摘要(Abstract)：

目的:探讨人脐静脉血管内皮细胞是否可作为饲养层来支持胚胎干细胞的生长,并维持其未分化状态。方法:使用健康产妇剖宫产后遗弃的脐带培养内皮细胞,并进行细胞传代培养和增殖扩增。将脱离饲养层克隆生长良好的E14.1胚胎干细胞接种到经丝裂霉素C(10mg/L)灭活后的2～5代内皮细胞饲养层上进行传代培养,对培养的胚胎干细胞进行碱性磷酸酶、Oct-4及体内全能分化实验鉴定。结果:E14.1胚胎干细胞在人脐静脉内皮细胞上传3～8代后能较好地保持未分化状态,高度表达碱性磷酸酶及干细胞因子Oct-4;体内全能分化实验显示,在内皮细胞上生长6代和10代以上的E14.1胚胎干细胞被接种到裸鼠6周后,形成含三个胚层的组织细胞畸胎瘤。结论:(1)人脐静脉血管内皮细胞可作为饲养层支持胚胎干细胞的生长;(2)胚胎干细胞在内皮细胞上能保持未分化状态;(3)人脐静脉血管内皮细胞可以作为一种来源丰富,取材方便的人源化饲养层细胞新来源,为胚胎干细胞的临床应用奠定了基础。
Objective:To explore whether umbilical vein endothelial cell (EC) can be used as feeder layers to support the growth of embryonic stem cells (ESC) and to retain their un-differentiation status. Methods:The abandoned umbilical cords were collected from healthy puerpera,and ECs were cultured and proliferated under asepsis condition. The morphologic characteristics of the cells were observed with inverted microscope and factor Ⅷ antigen was identified by using immunochemical method. The cultured cells from the second to the fifth generations were deactivated by treatment of mitomycin-C (10ug/ml) for 30～60 minutes,before E14.1 ESC colonies that isolated from feeder layer and were in good state were seeded on to them. The indexes used to indicate the living and differentiation state of the ESCs included alkaline phosphatase (AKP) activity,transcription factor Oct-4 expression,and the in vivo omnipotence test on the hind leg of nude mouse. Results:E14.1 ESCs formed good colonies,retained undifferentiated state and highly expressed a membrane alkaline phosphatase and a high level of Oct-4 when they were cultured and passed for 3~8 passages on human umbilical vein ECs. The E14.1 ESCs cultured on ECs for more than 6 passages and 10 passages formed teratomas containing three embryonic layers in 6 weeks after being inoculated into the hind leg of nude mice. Conclusions:ECs from allantoic veins can be used as feeder cells to support growth of ESCs. ESCs cultured on ECs can keep their undifferentiated characterization. Human umbilical vein ECs might become a rich source of feeder layers. The results of this study would lay a base for future clinical application.

关键词(KeyWords)： 干细胞;脐静脉;人;内皮,血管
stem cell; umbilical vein; persons; endothelium,vascular

Abstract:

Keywords:

基金项目(Foundation): 教育部新世纪优秀人才支持计划(NCET-05-0820);; 贵州省省长资金资助[黔科教办(2007)07号]

作者(Author): 王志华,何志旭,米蔷,汪浩文
WANG Zhihua,HE Zhixu,MI Qiang,WANG Haowen(Department of Pediatrics

DOI: 10.19367/j.cnki.1000-2707.2008.06.012

参考文献(References)：

• [1]Thomson JA,Itskoritz-Eldor J,Shaoiro SS,Waknitz MA,et al.Embryonic stem cell lines derived from humanblas-tocysts[J].Science,1998(5391):1145-1147.
• [2]Reubinoff BE,Pera MF,Fong C et al.Embryonic stem cell lines from human blastocysts:somatic differentiation in vitro[J].Nat Biotechnol,2000(4):399-404.
• [3]Qin Shen,Susan KG,Li Jin,et al.Endothelial cells stimulate self-renewal and expand neurogenesis of neural stem cells[J].Science,2004(5673):1338-1340.
• [4]牛青霞,何韶衡,陈韩秋.人脐静脉内皮细胞的体外培养与鉴定新方法探索[J].汕头大学医学院学报,2005(18):5-7.
• [5]David C.Hay,Linda Sutherland,John Clark,et al.Oct-4knockdown induces similar patterns of endoderm and troph-oblast differentiation markers in human and mouse embry-onic stem cells[J].Stem Cells,2004(2):225-235.
• [6]Richards M,Fong CY,Chan WK,et al.Human feeders support prolonged undifferentiated growth of human inner cell masses and embryonic stem cells[J].Nat Biotechn-ol,2002(9):933-936.
• [7]Cheng L,Hammond H,Ye Z,et al.Human adult mar-row cells support prolonged expansion of human embryonic stem cell in culture[J].Stem Cells,2003(2):131-142.
• [8]Suemori H,Tada T,Torii R,et al.Establishment of em-bryonic stem cell lines from cynomolgus monkey blasto-cysts produced by IVF or ICSI[J].Dev Dyn,2001(2):273-279.
• [9]Niwa H,Miyazaki J,Smith AG..Quantitative expression of Oct-3/4defines differentiations or self-renewal of ES cells[J].Mol Cell Biol.2002(5):1526-1536.
• [10]Tom Burdon,Davidc Hay,Linda Sutherland et al.Oct-4knockdown induces similar patterns of endoderm and trophoblast differentiation markers in human and mouse embryonic stem cells[J].Stem Cells,2004(2):225-235.
• [11]Guha Krishnaswamy,Jim Kelley,Lakshm Inarayanyerra,et al.Human endothelium source of multifunctional cytokines:molecular regulation and impossible role in human disease[J].Interferon and Cytokine Res,1999(1):91-96.
• [12]Broudy VC,Kaushansky K,Harlan JMet al.Interleukin1stimulates human endothelial cells to produce granulocyte-macrophage colony-stimulating factor and granulocyte colo-ny-stimulating factor[J].J Immunol,1997(2):464-468.

文章评论(Comment)：