大鼠羊膜细胞体外培养及其干细胞标记物的表达

Isolation,culture and identification of rat amniotic cells in vitro and their expression of stem cell markers

目的分离并鉴定大鼠羊膜细胞,探索羊膜细胞分化潜能,为干细胞移植治疗探寻新的细胞来源。方法机械分离羊膜组织并采用0.25%含EDTA的胰蛋白酶消化,应用高糖-DMEM培养基(添加10μg/L人表皮生长因子)培养,采用流式细胞术检测间充质干细胞表面标志物,通过细胞免疫荧光染色检测神经干细胞表面标记物。结果大鼠羊膜组织可分离并培养,细胞呈梭型,贴壁生长,短期内可以稳定增殖。细胞表达干细胞表面标记物八聚体结合转录因子4(octamer-binding transcription factor,Oct-4)和性别决定区Y框蛋白(sex determining region Y box 2,Sox-2),间充质干细胞表面标志物vimentin,胚胎干细胞标记物阶段特异性胚胎表面标记物(stage specific embryonic antigen-4,SSEA-4),并表达神经干细胞表面标志物nestin,可表达脑源性神经营养因子(brain derived neurotrophic factor,BDNF)和神经生长因子(nerve growth factor,NGF)。结论大鼠羊膜细胞可表达间充质干细胞和神经干细胞标记物,并有神经营养因子表达,为大鼠羊膜细胞的研究和应用提供实验依据。

Objective To isolate and identify the rat amniotic cells and to explore their stem cell characteristics, so as to find a new cell source for stem cell transplantation.Methods Rat amnion tissue was mechanically separated from 18-18.5-day old SPF pregnant rats and 0.25%trysin-EDTA digestion was used to obtain amniotic cells.The isolated cells were cultured with DMEM D-glucose added with 10 μg/L EGF.Flow cytometry was used to detect the mesenchyme stem cellsurface markers.Neural stem cell surface markers of the rat amniotic cells were detected using immunofluorescence staining.Results The rat amniotic membrane tissue was separated and mesenchymal stem cells were cultured successfully.The cells were ameboid-shaped and showed adherent growth, and can stably proliferate in short term.After culture, the cells expressed stem cell markers e.g.Oct-4 and Sox-2, mesenchymal stem cell markers e.g.vimentin, embryonic stem cell markers e.g.SSEA-4, neural stem cell marker e.g.nestin, and could also express neurotrophic factors, such as BDNF and NGF.Conclusions Rat amniotic cells express mesenchymal stem cells markers, neural stem cell markers and neurotrophic factors, therefore, provide an experiment basis for further research and application of rat amniotic cells in stem cell transplantation.