本文選題：羊膜 + 人羊膜上皮細(xì)胞　； 參考：《遵義醫(yī)學(xué)院》2012年碩士論文

【摘要】：目的：成體干細(xì)胞(Somatic stem cells, SSCs)移植作為治療神經(jīng)系統(tǒng)疾病新方法頗受關(guān)注,本研究旨在通過體外誘導(dǎo)實(shí)驗(yàn),評價(jià)人羊膜干細(xì)胞(Human amnion-derived stem cells, hAD-SCs)向神經(jīng)細(xì)胞(Neural Cells, NCs)分化的能力,為hAD-SCs作為治療神經(jīng)系統(tǒng)疾病新的細(xì)胞供源提供實(shí)驗(yàn)依據(jù)。 方法：①細(xì)胞分離及鑒定：采用胰酶-膠原酶兩步消化法從足月分娩的人羊膜組織中分離人羊膜上皮細(xì)胞(human amniotic epithelial cells, hAECs)和人羊膜間充質(zhì)干細(xì)胞(human amnion-derived mesenchymal stem cells, hAD-MSCs),用流式細(xì)胞儀(Flow cytometry, FCM)和免疫細(xì)胞化學(xué)(Immunocytochemistry, IC)法進(jìn)行表型鑒定和細(xì)胞鑒定。②誘導(dǎo)培養(yǎng)條件：hAECs和hAD-MSCs均設(shè)誘導(dǎo)組和未誘導(dǎo)組。誘導(dǎo)組為含200mM L-Glu和1μmol/L全反式維甲酸(All-trans-retinoic acid, ATRA)的無血清HG-DMEM培養(yǎng)基,未誘導(dǎo)組為含200mM L-Glu和10%胎牛血清HG-DMEM培養(yǎng)基。在37℃,5%CO2,飽和濕度條件下培養(yǎng)48h或96h。③NSE陽性細(xì)胞百分率及神經(jīng)元標(biāo)志物檢測：采用FCM分別檢測hAECs和hAD-MSCs誘導(dǎo)后NSE陽性細(xì)胞百分率；采用IC和免疫熒光染色(immunofluorescence, IF)法檢測神經(jīng)元特異性烯醇化酶(neuron-specific enolase, NSE)、神經(jīng)絲(neurofilament, NF)、膠質(zhì)纖維酸性蛋白質(zhì)(glial fibrillary acidic protein, GFAP)、(?)(?)經(jīng)微管蛋白(p-tubulin-Ⅲ)、微管結(jié)合蛋白2(microtubule-associated protein-2, MAP-2)及神經(jīng)元核蛋白(Neuronal Nuclei, NeuN)的表達(dá)。實(shí)時聚合酶鏈反應(yīng)(Real time polymerase chain reaction, RT-PCR)檢測NSE、NF、GFAP、β-tubulin-Ⅲ、MAP-2及NeuNmRNA的表達(dá)。④采用酶聯(lián)免疫吸附試驗(yàn)(ELISA)檢測hAECs和hAD-MSCs培養(yǎng)上清液中多巴胺(DA)的含量。 結(jié)果：①FCM和IC分析結(jié)果顯示(?)AECs CK19表達(dá)陽性,低表達(dá)CD44,不表達(dá)CD71、CD34、CD45;而hAD-MSCs高表達(dá)CD44、CD29、CD90、CD73、CD105及波形蛋白陽性,不表達(dá)CD34、CD45、CD19、CD14和HLA-DR。②AECs和hAD-MSCs誘導(dǎo)組細(xì)胞表達(dá)NSE百分率分別為61.16±19.8%和47.03±19.2%,而未誘導(dǎo)組幾乎不表達(dá)。hAECs和hAD-MSCs誘導(dǎo)組表達(dá)NSE、NF、GFAP、β-tubulin-Ⅲ,MAP-2及NeuN,而其未誘導(dǎo)組未見表達(dá)。hAECs和hAD-MSCs誘導(dǎo)組NSE、NF、 GFAP、β-tubulin-Ⅲ、MAP-2及NeuNmRNA的表達(dá)均高于未誘導(dǎo)組(P0.05)③hAECs和hAD-MSCs誘導(dǎo)組上清液中DA含量分別為79.71+11.94n∥L和74.03±9.46n∥L,明顯高于未誘導(dǎo)組(P0.05)。 結(jié)論：hAECs和(?)hAD-MSCs均具有向NCs分化的能力,提示hAD-SCs可作為治療神經(jīng)系統(tǒng)疾病新的細(xì)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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