【作者】 林芳；

【导师】 孟春；

【作者基本信息】 福州大学 ， 生物化学与分子生物学， 2014， 硕士

【摘要】 目的：建立高效稳定的饲养层培养体系用于小鼠胚胎干细胞的分离培养；鉴定E14TG2a小鼠胚胎干细胞的生物学特性并激活其Wnt/β-catenin信号通路,探究该信号通路的激活对小鼠胚胎干细胞向造血干/祖细胞定向分化的调控作用。方法：制备小鼠胚胎成纤维细胞作为饲养层细胞,用MTT法探究丝裂霉素C的最佳作用时间及浓度,从而建立高效稳定的胚胎干细胞分离培养体系；碱性磷酸酶(Alkaline phosphatase, AP)染色、细胞周期检测及核型分析鉴定E14TG2a小鼠胚胎干细胞的生物学特性；用外源性蛋白wnt3a(100ng/ml)持续作用E14TG2a小鼠胚胎干细胞21d,通过细胞免疫荧光及蛋白免疫印迹检测细胞内β-catenin蛋白含量,qRT-PCR检测Wnt信号通路下游靶标基因的表达量来确定是否激活Wnt/β-catenin信号通路；采用流式细胞仪检测造血干细胞表面标志CD34+/Sca-1+,同时用qRT-PCR检测造血相关基因的表达量,探究E14TG2a小鼠胚胎干细胞的Wnt/β-catenin信号通路激活后能否促进其向造血干细胞分化。结果：1、成功建立了高效稳定的小鼠胚胎干细胞饲养层培养体系。2、培养在饲养层上的E14TG2a小鼠胚胎干细胞在培养过程中基本保持未分化状态,细胞增殖活跃,胞内染色体没有出现异常的缺失、扩增或移位。3、E14TG2a小鼠胚胎干细胞经外源性蛋白wnt3a (100ng/ml)持续作用21d后,发现β-catenin蛋白在细胞内的含量增多；Wnt信号通路的下游靶标基因Pitx2、 Frizzled、Sox17、Oct4的表达量均出现不同程度的增加。4、E14TG2a小鼠胚胎干细胞的Wnt/β-catenin信号通路激活后,诱导其向造血干细胞分化。检测结果表明CD34+/Sca-1+细胞含量显著高于对照组,造血相关基因BMP4、FLK2及CD34的表达量均增加,而抑制造血分化的基因Smad5的表达则明显受到抑制。结论：E14TG2a小鼠胚胎干细胞经外源蛋白wnt3a (100ng/ml)持续作用21天后Wnt/β-catenin信号通路被激活,并促进其向造血干细胞的定向分化。更多还原

【Abstract】 Objects:In this work, we aimed to obtain a stable and efficient feeding layer cell to culture mouse embryonic stem cells, identify the characteristics of E14TG2a mouse embryonic stem cells.The regulation of cell differentiation mediated by Wnt/β-catenin signal pathway activation from mouse embryonic stem cells to hematopoietic stem/progenitor cells was also investigated.Methods:The feeder layer cells were prepared with mouse embryonic fibroblast. Mitomycin C concentrations in culture medium were determined by MTT to establish a highly efficient and stable mouse embryonic stem cell feeding layer cultivation system. The AP staining, cell cycle detection and karyotype analysis were performed to identify the characteristics of the E14TG2a mouse embryonic stem cells. The β-catenin level in E14TG2a cells was tested by cell immunofluorescence and western blot after treated with exogenous protein wnt3a (100ng/ml) for 21 days, and the expression of downstream target gene of Wnt signal pathway was detected by qRT-PCR to investigate whether the pathway was activated. To explore the effect of Wnt/β-catenin signaling pathway activation, the hematopoietic stem cell surface markers CD34+/Sca-1+ were detected by flow cytometry (FCM), and the expression of hematopoietic associated genes were measured by qRT-PCR.Results:1. We successfully establish a stable and efficient feeding layer cell to culture mouse embryonic stem cells.2. The E14TG2a mouse stem cells maintain undifferentiated state and high cell proliferation activity; its chromosomes did not show abnormity including deletions, amplification or shift.3. The β-catenin level in E14TG2a mouse stem cells is up-regulated after treated with wnt3a (100ng/ml) for 21 days. The expression of Wnt signal pathway’s downstream target genes such as Pitx2, Frizzled, Sox17 and Oct4 showed significant increase.4. After activating the Wnt/β-catenin signal pathway we induce E14TG2a mouse stem cells to differentiate to hematopoietic stem cells. The number of CD34+/Sca-1+ cells is significantly higher than that of the control group. The expression of hematopoietic associated genes BMP4, FLK2 and CD34 increases while the hematopoietic differentiation inhibitor gene SmadS is suppressed.Conclusion:Our data suggest that the activation of Wnt/β-catenin signal pathway could promote the directional differentiation of E14TG2a mouse stem cells to hematopoietic progenitor cells by treated with exogenous wnt3a (100ng/ml) for 21 days.更多还原