小鼠胚胎成纤维细胞滋养层对小鼠诱导多能干细胞的作用研究

摘要

目的:探讨小鼠胚胎成纤维细胞(mouse embryonic fibroblasts,MEFs)滋养层对小鼠诱导多能干细胞(induced pluripotent stem cells,iPSCs)适宜的培养条件及其作用机制.方法:取E12.5~14.5d ICR孕鼠,培养小鼠胚胎成纤维细胞(mouse embryonic fibroblasts,MEFs),制备滋养层,收集第2~3代(P2~P3)和第6代(P6)培养2~4d MEFs滋养层细胞培养基(MEF-CM),ELISA检测P2~P3和P6 MEF-CM中Activin A、白血病抑制因子(leukemia inhibitory factor,LIF)的浓度水平.iPSCs与滋养层细胞共同培养,并进行细胞鉴定.结果:ELISA检测结果显示P2~P3 MEF-CM中Activin A、LIF的浓度显著高于P6 MEF-CM,差异有显著性意义(P<0.05).iPSCs呈克隆状生长,细胞鉴定结果显示:拟胚体形成;碱性磷酸酶染色呈阳性;0CT4表达阳性.结论:E12.5~14.5d来源的P2~P3 MEFs滋养层能有效的抑制iPSCs的分裂,支持iPSCs的生长并维持其全能性.%Objective To establish mouse embryonic fibroblasts (MEFs) as the feeder layers for supporting induced pluripotent stem cells (iPSCs) and to discuss the effects of MEFs on iPSCs. Methods Mouse embryonic fibroblast cells for primary culture were derived from ICR mouse embryos (pregnant 12.5-14.5 days). MEFs were treated with mitomycin-C by 10ug/ml as feeder layer. MEF-conditioned medium (MEF-CM) was collected from the second or third and the sixth passage of MEFs. The concentration of Activin A and leukemia inhibitory factor (LIF) in MEF-CM was detected by enzyme-linked immunosorbent assay (ELISA). iPSCs were cultured on MEFs. Theexpression of alkaline phosphatase (ALP) and Octamer-4(OCT4) of iPSCs was tested. EB formation was achieved. Results After being treated with mitomycin-C,MEFs proliferation could be effectively repressed and be made into the feeder layer for iPSCs clonal expansion. ELISA was shown that the concentration of Activin A and LIF in the third passage of MEF-CM was significantly higher than that in the sixth passage of MEF-CM(P<0.05). iPSCs cultured on the feeder layer grew well and maintained undifferentiation and vitality, which could form the "nest" morphology as embryonic stem cells clone, and also formed EB. iPSCs expressed the positive result of ALP and OCT4. Conclusion MEF feeder layer derived from mouse embryos (E12.5-14.5d) could effectively support mouse iPSCs undifferentiation and self-renewal.