诱导性多潜能干细胞滋养层制备条件的实验研究

Study on preparation of feeder layers for induced pluripotent stem cells

目的制备小鼠胚胎成纤维细胞(Mouse Embryonic Fibroblasts,MEFs)滋养层,用于诱导性多潜能干细胞(Induced Pluripotent Stem Cells,iPSCs)的体外培养。方法取13.5～15.5天胎龄的胎鼠,采用组织消化法分离原代培养成纤维细胞,对MEFs的生长形态、生长曲线进行观察。采用10μg/ml丝裂霉素C预处理P2～P3代MEFs 2～3h制备出细胞滋养层,在该滋养层上进行iPSCs培养,观察iPSCs的集落生成情况,并行碱性磷酸酶细胞染色。结果 MEFs经丝裂霉素C预处理后细胞既不增殖,也不会死亡,仍保持分泌多种生长因子的能力。iPSCs在MEFs滋养层上生长良好,类似于胚胎干细胞一样能形成"鸟巢"状集落,并可维持iPSCs正常形态,抑制其分化而不影响活力,碱性磷酸酶染色阳性。结论利用诱导性多潜能干细胞滋养层方法制备的细胞滋养层,能有效支持iPSCs的生长,为进一步开展iPSCs的研究提供了理论依据和实验基础。

Objective To establish mouse embryonic fibroblasts（MEFs） as the feeder layers for supporting induced pluripotent stem cells（iPSCs） in vitro.Methods The mouse primary embryonic fibroblasts were isolated from the mouse embryos of 13.5-15.5 day gestation through primary tissue digestion.The morphology and growth curve of MEFs in vitro was investigated.And then the second or third passage of MEFs was treated with 10μg/ml mitomycin C for 2-3 hours to prepare fibroblast feeder layers.IPSCs were cultured on the feeder layer and iPSCs growth was observed.The expression of alkaline phosphatase（AKP） of iPSCs was tested.Results After treated by mitomycin C,MEFs had the capability to secrete multiple growth factors,neither proliferation nor apoptosis.iPSCs were cultured on the feeder layer grew well and maintained undifferentiation and vitality,which also could form the ＂nest＂ morphology as embryonic stem cells clone and show the positive result of AKP.Conclusion Preparing MEFs as feeder layers with the measure mentioned about can be used for culturing iPSCs in vitro.