The purpose of this study is to observe the effect of icariside II (ICS II) on the differentiation of human amniotic mesenchymal stem cells (hAMSCs) into dopaminergic neuron-like cells, the involvement of PI3K signaling pathway inhibitors. After identifying hAMSCs by flow cytometry, hAMSCs were induced and treated with ICS II at 10 μmol/L, 3 μmol/L, 1 μmol/L, and 0 μmol/L. hAMSCs in the LY294002+3μM ICS II group were pretreated with 20 μmol/L LY294002, a PI3K-specific inhibitor, for 1 h, and then hAMSCs were induced with 3 μmol/L ICS II. On the 21st day of induction, immunofluorescence was used to detect expression of the neuronal nuclei (NeuN), neuron-specific enolase (NSE), microtubule-associated protein-2 (MAP-2), glial fibrillary acidic protein (GFAP), and tyrosine hydroxylase (TH) antigens in each induced cell group. Western blotting was used to detect the relative protein expression of NSE, MAP-2, GFAP, and TH. ELISA was used to detect the dopamine concentration in the induction medium supernatant of each group. After 21 d of ICS II induction, immunofluorescence showed that GFAP expression was not obvious in any hAMSC group. The NeuN, NSE, MAP-2, and TH fluorescent proteins were expressed in each group. NeuN was expressed in the nucleus and cytoplasm, while NSE, MAP-2, and TH were mainly expressed in the cytoplasm. The positive cell rates of NeuN, NSE, MAP-2, and TH in the 10 μmol/L, 3 μmol/L, and 1 μmol/L ICS II groups were higher than those in the LY294002+3μM ICS II and control groups. After 21 d of induction, the Western blot results showed that the protein expression levels of NSE, MAP-2, and TH in the 10 μmol/L, 3 μmol/L, and 1 μmol/L ICS II groups were significantly higher than those in the LY294002+3μM ICS II and control groups. The MAP-2 protein expression levels in the 10 μmol/L and 3 μmol/L groups were higher than that in the 1 μmol/L group. After 21 d of induction, the dopamine concentrations in the culture supernatants of the 10 μmol/L, 3 μmol/L, and 1 μmol/L ICS II groups were higher than those in the LY294002+3μM ICS II and control groups. In our experiment, ICS II induced hAMSCs to differentiate into dopaminergic neuron-like cells, and the optimal concentration range of ICS II was 3–10 μmol/L. Moreover, the PI3K signaling pathway is involved in the above differentiation process.

本研究的目的是观察淫羊藿苷II（ICS II）对人羊膜间充质干细胞（hAMSCs）向多巴胺能神经元样细胞分化的影响，PI3K信号通路抑制剂的参与。通过流式细胞术鉴定 hAMSCs 后，用 10 μmol/L、3 μmol/L、1 μmol/L 和 0 μmol/L 的 ICS II 诱导和处理 hAMSCs。LY294002+3μM ICS II组hAMSCs用20 μmol/L LY294002（一种PI3K特异性抑制剂）预处理1 h，然后用3 μmol/L ICS II诱导hAMSCs。在诱导第 21 天，使用免疫荧光检测神经元核 (NeuN)、神经元特异性烯醇化酶 (NSE)、微管相关蛋白-2 (MAP-2)、胶质纤维酸性蛋白 (GFAP) 和每个诱导细胞组中的酪氨酸羟化酶 (TH) 抗原。Western blotting检测NSE、MAP-2、GFAP和TH的相对蛋白表达。采用ELISA检测各组诱导培养基上清液中多巴胺浓度。ICS II诱导21 d后，免疫荧光显示GFAP在任何hAMSC组中表达均不明显。NeuN、NSE、MAP-2 和 TH 荧光蛋白在每组中均有表达。NeuN在细胞核和细胞质中表达，而NSE、MAP-2和TH主要在细胞质中表达。10 μmol/L、3 μmol/L和1 μmol/L ICS II组NeuN、NSE、MAP-2和TH阳性细胞率高于LY294002+3 μM ICS II组和对照组。诱导21 d后，Western blot结果显示NSE、MAP-2、TH的蛋白表达水平分别在10 μmol/L、3 μmol/L、和1 μmol/L ICS II组显着高于LY294002+3μM ICS II组和对照组。10 μmol/L和3 μmol/L组MAP-2蛋白表达水平高于1 μmol/L组。诱导21 d后，10 μmol/L、3 μmol/L和1 μmol/L ICS II组培养上清中多巴胺浓度高于LY294002+3 μM ICS II组和对照组。在我们的实验中，ICS II诱导hAMSCs分化为多巴胺能神经元样细胞，ICS II的最佳浓度范围为3-10 μmol/L。此外，PI3K信号通路参与了上述分化过程。10 μmol/L和3 μmol/L组MAP-2蛋白表达水平高于1 μmol/L组。诱导21 d后，10 μmol/L、3 μmol/L和1 μmol/L ICS II组培养上清中多巴胺浓度高于LY294002+3 μM ICS II组和对照组。在我们的实验中，ICS II诱导hAMSCs分化为多巴胺能神经元样细胞，ICS II的最佳浓度范围为3-10 μmol/L。此外，PI3K信号通路参与了上述分化过程。10 μmol/L和3 μmol/L组MAP-2蛋白表达水平高于1 μmol/L组。诱导21 d后，10 μmol/L、3 μmol/L和1 μmol/L ICS II组培养上清中多巴胺浓度高于LY294002+3 μM ICS II组和对照组。在我们的实验中，ICS II诱导hAMSCs分化为多巴胺能神经元样细胞，ICS II的最佳浓度范围为3-10 μmol/L。此外，PI3K信号通路参与了上述分化过程。ICS II诱导hAMSCs分化为多巴胺能神经元样细胞，ICS II的最佳浓度范围为3-10 μmol/L。此外，PI3K信号通路参与了上述分化过程。ICS II诱导hAMSCs分化为多巴胺能神经元样细胞，ICS II的最佳浓度范围为3-10 μmol/L。此外，PI3K信号通路参与了上述分化过程。