体外诱导人羊膜细胞向神经细胞分化

发布时间：2018-07-07 16:24

  本文选题：羊膜 + 人羊膜上皮细胞　； 参考：《遵义医学院》2012年硕士论文

【摘要】：目的：成体干细胞(Somatic stem cells, SSCs)移植作为治疗神经系统疾病新方法颇受关注,本研究旨在通过体外诱导实验,评价人羊膜干细胞(Human amnion-derived stem cells, hAD-SCs)向神经细胞(Neural Cells, NCs)分化的能力,为hAD-SCs作为治疗神经系统疾病新的细胞供源提供实验依据。 方法：①细胞分离及鉴定：采用胰酶-胶原酶两步消化法从足月分娩的人羊膜组织中分离人羊膜上皮细胞(human amniotic epithelial cells, hAECs)和人羊膜间充质干细胞(human amnion-derived mesenchymal stem cells, hAD-MSCs),用流式细胞仪(Flow cytometry, FCM)和免疫细胞化学(Immunocytochemistry, IC)法进行表型鉴定和细胞鉴定。②诱导培养条件：hAECs和hAD-MSCs均设诱导组和未诱导组。诱导组为含200mM L-Glu和1μmol/L全反式维甲酸(All-trans-retinoic acid, ATRA)的无血清HG-DMEM培养基,未诱导组为含200mM L-Glu和10%胎牛血清HG-DMEM培养基。在37℃,5%CO2,饱和湿度条件下培养48h或96h。③NSE阳性细胞百分率及神经元标志物检测：采用FCM分别检测hAECs和hAD-MSCs诱导后NSE阳性细胞百分率；采用IC和免疫荧光染色(immunofluorescence, IF)法检测神经元特异性烯醇化酶(neuron-specific enolase, NSE)、神经丝(neurofilament, NF)、胶质纤维酸性蛋白质(glial fibrillary acidic protein, GFAP)、(?)(?)经微管蛋白(p-tubulin-Ⅲ)、微管结合蛋白2(microtubule-associated protein-2, MAP-2)及神经元核蛋白(Neuronal Nuclei, NeuN)的表达。实时聚合酶链反应(Real time polymerase chain reaction, RT-PCR)检测NSE、NF、GFAP、β-tubulin-Ⅲ、MAP-2及NeuNmRNA的表达。④采用酶联免疫吸附试验(ELISA)检测hAECs和hAD-MSCs培养上清液中多巴胺(DA)的含量。 结果：①FCM和IC分析结果显示(?)AECs CK19表达阳性,低表达CD44,不表达CD71、CD34、CD45;而hAD-MSCs高表达CD44、CD29、CD90、CD73、CD105及波形蛋白阳性,不表达CD34、CD45、CD19、CD14和HLA-DR。②AECs和hAD-MSCs诱导组细胞表达NSE百分率分别为61.16±19.8%和47.03±19.2%,而未诱导组几乎不表达。hAECs和hAD-MSCs诱导组表达NSE、NF、GFAP、β-tubulin-Ⅲ,MAP-2及NeuN,而其未诱导组未见表达。hAECs和hAD-MSCs诱导组NSE、NF、 GFAP、β-tubulin-Ⅲ、MAP-2及NeuNmRNA的表达均高于未诱导组(P0.05)③hAECs和hAD-MSCs诱导组上清液中DA含量分别为79.71+11.94n∥L和74.03±9.46n∥L,明显高于未诱导组(P0.05)。 结论：hAECs和(?)hAD-MSCs均具有向NCs分化的能力,提示hAD-SCs可作为治疗神经系统疾病新的细胞供源。
[Abstract]:Objective: as a new method for the treatment of nervous system diseases, transplantation of human stem cells (SSCs) has attracted much attention. The aim of this study was to evaluate the ability of human amnion-derived stem cells (hAD-SCs) to differentiate into neural cells (NCs) through in vitro induction experiments. To provide experimental evidence for hAD-SCs as a new cell donor for the treatment of nervous system diseases. Methods: human amniotic epithelial cells (human amniotic epithelial cells, hAECs) and human amniotic mesenchymal stem cells (human amnion-derived mesenchymal stem cells, hAD-MSCs) were isolated from human amniotic membrane tissue by trypsin collagenase two-step digestion method. Flow cytometry was used to isolate human amniotic mesenchymal stem cells (human amnion-derived mesenchymal stem cells, hAD-MSCs). The phenotypic identification and cell identification were performed by flow cytometry (FCM) and immunocytochemistry (IC). 2. The induced culture conditions were as follows: hAECs and hAD-MSCs were divided into two groups: induced group and uninduced group. The induction group was serum-free HG-DMEM medium containing 200mm L-Glu and 1 渭 mol / L all-trans retinoic acid (ATRA), while the non-induction group was HG-DMEM medium containing 200mm L-Glu and 10% fetal bovine serum (HG-DMEM). The percentage of NSE positive cells and neuronal markers were detected by FCM after 48 h or 96 h. 3 NSE positive cells were cultured under saturated humidity. The percentage of NSE positive cells induced by hAECs and hAD-MSCs were detected by FCM. Neuron-specific enolase (NSE), neurofilamentase (NF), glial fibrillary acidic protein (glial fibrillary acidic protein, GFAP), (?) and glial fibrillary acidic protein (glial fibrillary acidic protein, GFAP), (?) were detected by IC and immunofluorescence staining (if). The expression of p-tubulin- 鈪，

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