大鼠胚胎上胚层干细胞的分离培养研究

Isolation and cultivation of epiblast stem cells from SD rat embryo

目的获取SD大鼠胚胎中附植后的胚胎上胚层干细胞(Epi SCs),并就影响SD大鼠Epi SCs(SDEpi SCs)分离与培养的组织来源、胎龄、细胞因子等条件进行探讨,从而探索适宜SD大鼠Epi SCs分离培养的实验体系,为下一步SD大鼠Epi SCs的建系提供实验研究基础。方法以昆明小鼠胚胎成纤维细胞为饲养层,分别取5.5日胎龄的囊胚和7.5日胎龄囊胚上胚层(Epiblast),用添加白血病抑制因子(LIF)或活化素(activin)的基础培养基进行原代培养,分别采用机械法和胰酶消化法对克隆集落进行传代培养。最后通过碱性磷酸酶染色对克隆集落进行鉴定分析,并检测其体外分化能力。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SDEpi SCs原代集落。经机械法传代获得AKP染色阴性、能形成拟胚体的SDEpi SCs。结论利用细胞因子activin,结合机械传代法可从7.5日胎龄SD大鼠胚胎中获得SDEpi SCs。

Objective To derive post-implantation epiblast stem cells（Epi SCs） from SD rat embryo, explore the effect of tissue origin, fetal age, and cytokines on the isolation and cultivation of SDEpi SCs, and investigate the suitable culture system for the growing of SD rat Epi SCs for the next step of the establishment of SD rat Epi SCs lines. Methods The whole blastocyst and epiblast were dissociated from embryo at fetal age of 5.5 day and 7.5 day and explanted on the mouse embryonic fibroblast（MEF） in the basic medium supplemented with leukemia inhibitory factory（LIF） or activin.The trypsin or mechanically dissociation was used for cell passaging. The characteristic of SDEpi SCs was analyzed by alkaline phosphatase staining. In vitro differentiation potential of SDEpi SCs was explored by the induction of embryo body.Results Severe spontaneous differentiation took place in the blastocysts cultured in LIF medium or activin medium, as well as the epiblast cultured in LIF medium. Epi SCs primary colonies emerged from the epiblast cultured in the activin medium. After passaging the colonies via mechanically dissociation, the alkaline phosphatase negative SDEpi SCs were obtained and showed the ability to form embryo body in vitro. Conclusion Combination of activin and mechanically dissociation, SDEpi SCs can be obtained from the SD embryo at fetal age 7.5 day.