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目的从SD大鼠胚胎中获取附植后胚胎上胚层干细胞(post-implantation epiblast stem cells,Episc),并探讨组织来源、胎龄、细胞因子、传代方法等条件对SD大鼠Episc(SD Episc)分离与培养的影响。方法分别取5.5d和7.75d胎龄囊胚上胚层(epiblast),以昆明小鼠胚胎成纤维细胞为饲养层,于添加白血病抑制因子(leucocyte inbibitory factor,LIF)或活化素(activin)的基础培养基中,进行原代培养。分别采用机械法和胰酶消化法对克隆集落进行传代。最后通过碱性磷酸酶染色对克隆集落进行鉴定分析,并对其体外分化能力进行检测。结果培养于LIF培养基和activin培养基中的囊胚均发生严重分化。培养于LIF的培养基中的epiblast也发生了严重的自发分化。而在activin培养基中,epiblast形成SD Episc原代集落。经机械法传代获得AKP染色阴性、能形成拟胚体的SD Episc。结论利用细胞因子activin,结合机械传代法可从7.75d胎龄SD大鼠胚胎中获得SD Episc。
OBJECTIVE: To obtain Episc (Ep Eps) of epididymal grafts from SD rat embryos and to investigate the effect of Episc (SD Episc) Effects of isolation and culture. Methods The embryonic fibroblasts from Kunming mice were used as the feeder layer for 5.5 days and 7.75 days gestational age embryos, respectively, on the basis of addition of leukemic inbibitory factor (LIF) or activin Medium, primary culture. Clonal colonies were passaged mechanically and trypsin digestion, respectively. Finally, the colonies were identified by alkaline phosphatase staining and their differentiation ability was tested. Results The blastocysts cultured in LIF medium and activin medium all had severe differentiation. Epiblast cultured in LIF medium also developed severe spontaneous differentiation. In the activin medium, epiblast formed SD Episc primary colonies. AKP stained negative by mechanical transmission and can form SD Episc of embryoid body. Conclusion SD Episc can be obtained from 7.75 d gestational age SD rat embryos using the cytokine activin combined with mechanical passaging.