不同滋养层对维持人胚胎干细胞的生长作用  

Different Types of Feeder Cells for Maintenance of Human Embryonic Stem Cells

作　　者：朱宛宛[1,2] 李宁[1,3] 王芳[1,2] 付琳琳[1,2] 徐艳玲[1,2] 关云谦[1,2] 张愚[1,2] 

机构地区：[1]首都医科大学宣武医院老年病研究所细胞治疗室,北京100053 [2]教育部神经变性病重点实验室,北京100053 [3]首都师范大学生命科学学院生殖与发育研究室,北京100037

出　　处：《中国医学科学院学报》2009年第4期468-472,I0004,I0005,共7页Acta Academiae Medicinae Sinicae

基　　金：国家重点基础研究发展计划(973项目)(2006CB0F0603;2007CB947700);北京市科学技术委员会科技计划资助项目(H020220010290)~~

摘　　要：目的比较不同滋养层对保持连续传代培养的人胚胎干细胞(hESC)不分化和高增殖能力作用的差异。方法分别用小鼠胚胎成纤维细胞(MEF)、人包皮成纤维细胞(hFF)、人骨髓间充质细胞(hMSC)作为滋养层培养hESC系H9细胞,采用碱性磷酸酶染色和台盼蓝染色观察不同滋养层支持的hESC的碱性磷酸酶阳性克隆数、细胞产量和细胞存活率,并采用免疫荧光染色对传代5代以后的hESC进行生物学鉴定。结果3种滋养层细胞均可维持hESC呈克隆样生长,但是不同滋养层支持的克隆的形态差异大。3种滋养层细胞支持的hESC的大鼠抗阶段特异性胚胎抗原-3及碱性磷酸酶染色均呈阳性。MEF支持的hESC的碱性磷酸酶阳性克隆数、细胞产量和细胞存活率均显著高于人来源滋养层(均P<0.01)。hMSC支持的hESC的碱性磷酸酶阳性克隆数、细胞产量均显著高于hFF的hESC(均P<0.01)。结论3种滋养层细胞均可支持hESC的正常生长。MEF最有利于hESC的增殖,但是两种人来源滋养层细胞为去除动物源性的hESC培养体系奠定了基础。在维持hESC增殖方面,hMSC优于hFF。Objective To compare the effects of different types of feeder cells on supporting undifferentiation and high proliferation of human embryonic stem cells （hESC）. Methods hESC were seeded on mouse embryonic fibroblasts （MEF）,human marrow stromal cells （hMSC）,and human foreskin fibroblasts （hFF）,respectively. Colony number,cell quantity after digestion,and survival rate were observed by alkaline phosphatase （AP） staining and Trypan blue,and the biological properties of hESC after 5 passages were observed by immunofluorescence staining. Results Although all the three feeder layers could support the formation of hESC colonies and maintain pluripotency,the morphology of colonies on different feeder layers remarkably varied. The stage-specific embryonic antigen-3 and AP staining were positive on three types of feeders. The number of colonies,number of cells produced,and cell survival rates were significantly higher on MEF than on human feeder cells （P〈0.01）.Furthermore,the number of AP-positive colonies and cell quantity were also significantly higher on hMSC than on hFF （P〈0.01）. Conclusions All three types of feeder cells are able to support the growth of hMSC,although MEF are more favourable for the proliferation. Two types of human feeder cells lay the foundation for the removal of animal-derived hESC culture system. hMSC is superior to hFF in supporting the proliferation of hESC.

关 键 词：人胚胎干细胞 滋养层 包皮成纤维细胞 骨髓间充质干细胞 

分 类 号：Q254[生物学—细胞生物学]