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目的:探讨黄芪注射液体外定向诱导人羊膜上皮细胞分化的作用和黄芪注射液对分化细胞活力影响。方法:将人羊膜上皮细胞分为三组,即黄芪诱导组(设立四个浓度亚组)、全反式维甲酸组和对照组。分别采用化学诱导剂和黄芪注射液诱导hAECs分化为神经细胞,在诱导前后,应用免疫细胞化学方法染色鉴定神经元特异性烯醇化酶、神经干细胞巢蛋白、胶质纤维酸性蛋白.逆转录-聚合酶链反应进一步鉴定细胞多能基因Oct4、神经干细胞标记物Nestin基因和神经元标记物NSE,四甲基偶氮唑盐比色法观察细胞活力。结果:倒置显微镜下见,RA诱导12 h后,黄芪诱导24 h后,由胞体伸出较长的轴突样和树突样突起,且有分支,48 h时100μl/ml黄芪诱导组,NSE阳性率低于RA诱导组(P<0.05),细胞活力较RA组高(P<0.05),两诱导组结果均高于对照组,而GFAP阳性率高于RA组(P<0.05),同时RT-PCR检测到诱导后多能基因Oct4表达减少,而Nestin、NSE表达增多。结论:黄芪和RA都可诱导人羊膜上皮细胞在体外分化为神经元样细胞,且黄芪于100μl/ml浓度时效果较好,黄芪诱导后对细胞毒性较小。
OBJECTIVE: To investigate the effect of astragalus injection on the differentiation of human amniotic epithelial cells induced in vitro and the effect of Astragalus injection on the viability of differentiated cells. Methods: Human amniotic epithelial cells were divided into three groups, Astragalus induction group (four concentration subgroups), all-trans retinoic acid group and control group. The hAECs were induced to differentiate into neurons by chemical inducer and astragalus injection respectively, and the neuron-specific enolase, neural stem cell nestin and glial fibrillary acidic protein were identified by immunocytochemical staining before and after induction.Reverse transcription-polymerization Enzyme-linked reaction further identified cell pluripotency gene Oct4, neural stem cell marker Nestin gene and neuronal marker NSE, cell viability was observed by MTT assay. Results: Under inverted microscope, after 12 h of RA induction, astragalus was induced by astragalus for 24 h, then the axon-like and dendritic-like protuberances extended from the cell body and branches were formed. After 48 h, 100 μl / ml astragalus induction group, NSE The positive rate of GFAP in RA group was lower than that in RA group (P <0.05), and the viability of RA group was higher than that in RA group (P <0.05). The positive rates of GFAP in both groups were higher than those in RA group (P <0.05) RT-PCR detected a decrease of pluripotency gene Oct4 expression and increased expression of Nestin and NSE. Conclusion: Astragalus membranaceus and RA can induce human amniotic membrane epithelial cells to differentiate into neuron - like cells in vitro. Astragalus membranaceus is more effective in the concentration of 100μl / ml and less toxic to the astragalus after induction.