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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 investigate the effect of E1A-stimulated E1A-stimulated genes (CREG) on the self-renewal of embryonic stem cells (ESC) and its mechanism of action, this study aimed to establish a low expression of CREG by RNA interference Feeder STO cells, lay the foundation for further research. Methods: The expression of CREG gene in feeder cell line STO and ESC cell line R1 was detected by Western Blot. RNA interference empty vector was transfected into STO cells by Lipofectamine 2000, a control vector containing nonsense random sequence and a vector containing four different CREG interference sequences. Screening with 1.0μg / ml puromycin for 1 w, under the fluorescence microscope to select a higher expression of green fluorescent protein clone amplification. The Western Blot method was used to identify the interference efficiency of CREG gene and obtain the feeder cell clone with the lowest CREG expression. ESC R1 was inoculated to this clone, without adding leukemia inhibitory factor, cultured continuously for 3 passages, and its differentiation was judged by alkaline phosphatase staining. Results: Rl hardly expressed CREG, while STO cells highly expressed CREG. Western Blot results confirmed that the screening of STO clone 3A best interference effect, reaching 85%. In the absence of leukemia inhibitory factor, alkaline phosphatase staining showed that there was no obvious differentiation of R1 cells on the feeder layer of three generations. CONCLUSION: The successful obtaining of STO cells with low expression of CREG in feeder layer provides the basis for further study on the role and mechanism of CREG on ESC self-renewal.