摘要
碱性成纤维细胞生长因子(basic fi broblast growth factor,b FGF)在小鼠和人胚胎干细胞自我更新和分化过程中起着重要的调控作用,但目前关于bFGF在大鼠胚胎干细胞中的调控作用并不是很清楚。该文在无饲养层细胞的条件下,通过碱性磷酸酶染色、免疫荧光、RT-PCR等方法对大鼠胚胎干细胞在激活b FGF相关信号通路后自我更新、细胞分化潜能等进行了分析。结果显示,在白血病抑制因子(leukemia inhibitory factor,LIF)加GSK3抑制剂(CHIR99021)和ERK抑制剂(PD0325901)(简称L/2I)基础上,b FGF能明显促进大鼠胚胎干细胞的增殖。L/2I/b(L/2I+b FGF)条件下培养的大鼠胚胎干细胞能维持干性标志基因Oct-4、Nanog的表达,同时也保持了向不同胚层细胞分化的能力。另外,L/2I/b也能支持从大鼠囊胚原代分离胚胎干细胞。Western blot结果显示,b FGF能促进PI3K下游分子AKT的磷酸化。小分子化合物SU5402或LY294002分别抑制FGF受体及PI3K均能阻断b FGF激活的AKT磷酸化,并抑制b FGF对大鼠胚胎干细胞自我更新的促进作用。这些结果表明,b FGF通过激活大鼠胚胎干细胞PI3K/AKT相关的信号通路促进大鼠胚胎干细胞自我更新。
Basic fibroblast growth factor （bFGF） plays important roles in the control of pluripotency and lineage specification in mouse or human embryonic stem cells （ES cells） states. However, it is unclear whether bFGF signaling is involved in self-renewal of rat ES cells. To investigate the effect of bFGF on rat ES cells, rat ES cells were cultured in serum free medium supplemented with LIF, PD0325901 and CHIR99021 （L/2I） and with or without bFGF. Small molecular chemical SU5402 and LY294002 were used to inhibit FGF receptor and PI3K, respectively. Alkaline phosphatase staining, immunostaining and RT-PCR were performed to identify the pluripo- tency of rat ES cells. Western blot was used to analysis the phosphorylation level of AKT. The results showed that bFGF promoted self-renewal of rat ES cells under a feeder cell and serum free condition. Rat ES cells maintained with L/2I and bFGF expressed Oct-4 and Nanog and could differentiate to cells of ectoderm, mesoderm and endoderm. Furthermore, bFGF promoted the phosphorylation level of AKT and this effect could be inhibited by SU5402 or LY294002. Both SU5402 and LY294002 abolished the promoting effect of bFGF on the self-renewal of rat ES cells. In conclusion, our results indicated that bFGF promoted self-renewal of rat ES cells and this effect may mainly be regulated by PI3K/AKT dependent signaling pathway.
出处
《中国细胞生物学学报》
CAS
CSCD
2016年第5期550-556,共7页
Chinese Journal of Cell Biology
