黄芪诱导人羊膜上皮细胞WISH细胞株向神经细胞分化并抑制Notch1表达及促进细胞存活

Astragalus induces human amniotic epithelial cells (WISH) to differentiate toward neurons, inhibits the expression of Notch1 and promotes cell survival

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。

The aim of the study was to investigate the effect of astragalus on differentiation of human amniotic epithelial cell line WISH into neurons, the expression of Notch1 gene and cell viability. WISH were randomly divided into astragalus group （4 subgroups）, alltransretinoic acid （RA） group and control group. Astragalus group and RA group were induced to differentiate into neurocytes by using chemical inducer RA and astragalus, respectively. The expression of neuron-specific enolase （NSE）, microtubule associated protein 2 （MAP-2）, Nestin and GFAP of induced cells in three groups were detected using immunocytochemical method. RTPCR was further used to detect the expression of Oct4, Notch1, Hes1, Nestin and NSE. The cell viability was measured by methyl thiazolyl tetrazolium methods. Under the convert microscope it was observed that WISH cells started to change their shape, and there were several axon or dendrite-like processes out from the cell body induced by astragalus for 24 h or RA for 12 h. The positive cell rates of NSE and MAP-2 in 100 μL/mL astragalus-induced group were less than those in RA-induced group at 48 h （P 0.05）, but higher than those in control group. Cell viability in astragalus group was higher than that of RA group （P 0.05）. While the positivecell rates of Nestin and GFAP in 100 μL/mL astragalus-induced group were higher than those in RA-induced group at 48 h （P 0.05）. The positive cell rates of Nestin in the two induced groups were lower than those in control group. RT-PCR showed that the expressions of Oct4, Notch1 and Hes1 in RA and astragalus （100 μL/mL） groups were less than those in control group, but the expression of NSE was higher than that in control group. These results suggest that astragalus （especially at 100 μL/mL, 48 h） and RA can both induce human amniotic epithelial cell line WISH cells into neuron-like cells, but astragalus induction has a higher cell survival rate than RA induction, and the expression of Notch1 signal molecules is inhibited during the induction.