CREG低表达饲养层STO细胞的建立  

Establishment of CREG Knocked Down STO Feeder Cells

作　　者：田孝祥[1] 张剑[1] 陶杰[1] 孙鸣宇[1] 闫承慧[1] 韩雅玲[1] 

机构地区：[1]沈阳军区总医院全军心血管病研究所心内科,辽宁沈阳110840

出　　处：《现代生物医学进展》2012年第31期6034-6037,6010,共5页Progress in Modern Biomedicine

基　　金：国家自然科学基金重点项目(81130072);国家自然科学基金面上项目(81070097);国家科技部973前期项目(2011CB512111);辽宁省科技攻关项目(2010225036)

摘　　要：目的:为阐明E1A激活基因阻遏子(Cellular repressor of E1A-stimulated genes,CREG)对胚胎干细胞(Embryonic stem cell,ESC)自我更新的影响及作用机制,本研究拟通过RNA干扰方法建立CREG低表达的饲养层STO细胞,为深入研究奠定基础。方法:用Western Blot方法检测饲养层细胞系STO及ESC细胞系R1中CREG基因的表达。利用Lipofectamine 2000向STO细胞中分别转染RNA干扰空对照载体,含有无意义随机序列的对照载体以及含有4种不同CREG干扰序列的载体。用1.0μg/ml嘌呤霉素筛选1 w,荧光显微镜下挑取绿色荧光蛋白表达较高的克隆进行扩增。Western Blot方法鉴定CREG基因干扰效率,获得CREG表达最低的饲养层细胞克隆。将ESC R1接种到该克隆上,不添加白血病抑制因子,连续培养3代,用碱性磷酸酶染色判断其是否分化。结果:R1几乎不表达CREG,而STO细胞高表达CREG。Western Blot结果证实筛选到的STO克隆3A干扰效果最好,达到85%。在不添加白血病抑制因子的情况下,碱性磷酸酶染色表明R1细胞在该株饲养层细胞上连续培养3代后未见明显分化。结论:成功获得CREG低表达饲养层STO细胞,为深入探讨CREG对ESC自我更新的作用及机制奠定了基础。Objective： To clarify function and mechanism of CREG （Cellular repressor of E1A-stimulated genes） on ESC （Embryonic stem cell） self-renewal, we aimed to establish a feeder cell line STO with CREG knocked down by using RNA interference （RNAi） to pave the way for further investigation. Methods： Expression of CREG in feeder cell line STO and ESC cell line R1 was determined by Western Blot. One set of RNAi plasmids, including 1 empty negative control vector without insertion, 1 scrambled negative control vector containing non-effective sequence and 4 expression vectors with CREG-speciflc interfering sequence, were delivered to STO cells through Lipofectamine 2000 transfeetion. Transfected cells were then selected by 1.0 μg/ml puromycin for 1 week. Survived colonies expressing enhanced green fluorescence protein were picked up and expanded. CREG interference efficiency was assessed by Western Blot analysis and a feeder cell clone with lowest CREG expression level was obtained. R1 were cultured on this clone for 3 passages continuously with addition of leukemia inhibition factor and differentiation state of R1 was evaluated by alkaline phosphatase staining. Results： In contrast to R1 with few expression of CREG, STO cell showed significant high level of CREG expression. Western Blot analysis identified clone 3A of selected STO cells was the most effectively interfered in CREG expression with 85 % efficiency. Alkaline phosphatase staining showed that R1 had no significant sign of differentiation after 3 passages on CREG knocked down STO without addition of leukemia inhibition factor. Conclusion： We successfully establish a STO feeder cell line with CREG knocked down, which will pave the way for further investigating function and mechanism of CREG on ESC self-renewal.

关 键 词：CREG RNA干扰 饲养层细胞 胚胎干细胞 自我更新 

分 类 号：Q291[生物学—细胞生物学]