论文部分内容阅读
本研究旨在探讨黄芪(astragalus)对人羊膜上皮细胞WISH细胞株向神经细胞分化、Notch1基因表达和细胞活力的影响。将人羊膜上皮细胞WISH细胞株分为3组,即黄芪诱导组(设立4个浓度亚组)、全反式维甲酸(alltransretinoic acid,RA)组和对照组。分别采用化学诱导剂RA和黄芪注射液诱导WISH细胞株分化为神经细胞,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(neuron-specific enolase,NSE)、微管相关蛋白2(microtubule associated protein2,MAP-2)、神经干细胞巢蛋白(Nestin)、胶质纤维酸性蛋白(glial fibrillary acidic protein,GFAP)。逆转录-聚合酶链反应(RT-PCR)进一步鉴定细胞多能性基因Oct4、Notch1、Hes1、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(MTT法)观察细胞活力。倒置显微镜观察显示,WISH细胞株分别经RA诱导12h后、黄芪诱导24h后,由胞体伸出较长的轴突样和树突样突起,且有分支。诱导48h时,100μL/mL黄芪诱导组NSE和MAP-2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NSE、MAP-2和GFAP阳性率均高于对照组;而黄芪诱导组Nestin和GFAP阳性率高于RA组(P<0.05),两诱导组Nestin阳性率均低于对照组。同时RT-PCR检测到黄芪(100mL/mL)和RA诱导后多能基因Oct4、Notch1、Hes1表达低于对照组,而NSE表达增多。以上结果提示,黄芪和RA都可诱导WISH细胞株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,黄芪诱导对细胞毒性较小,诱导过程中能抑制Notch1信号分子的表达。
This study aimed to investigate the effects of astragalus on the differentiation of human amniotic epithelial cell line WISH into neuronal cells, Notch1 gene expression and cell viability. The human amniotic epithelial cells WISH cell lines were divided into three groups, namely Astragalus induction group (set up four concentration subgroups), all-trans retinoic acid (alltransretinoic acid, RA group and control group. WISH cells were induced to differentiate into neurons by chemical inducer RA and astragalus injection respectively. Immunocytochemical staining was used to identify neuron-specific enolase (NSE), microtubule-associated protein 2 (microtubule associated protein2, MAP-2), neural stem cell nestin and glial fibrillary acidic protein (GFAP). Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to further identify the pluripotency genes Oct4, Notch1, Hes1, Nestin gene, neural stem cell marker NSE, MTT France) observed cell viability. Inverted microscopy showed that after WISH cells were induced by RA for 12h, Astragalus membranaceus induced longer axonal-like and dendritic-like processes from the cell body and branching. At 48h after induction, the positive rate of NSE and MAP-2 in astragalus-induced group was lower than that in RA-induced group (P <0.05), and the viability of NSE, MAP-2 and GFAP The positive rates of Nestin and GFAP in astragalus induction group were higher than those in RA group (P <0.05). The positive rates of Nestin in two induction groups were lower than those in control group. At the same time, the expression of astragalus (100mL / mL) and pluripotency gene Oct4, Notch1 and Hes1 after RA induction were lower than those of the control group, while NSE expression increased. The above results suggest that both Astragalus and RA can induce WISH cell lines to differentiate into neuron-like cells in vitro, and Astragalus at 100 μL / mL concentration effect is better, astragalus induction of cytotoxicity is small, induction can inhibit Notch1 signaling molecules expression.