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背景:研究证明脐血间质干细胞可向神经元样细胞分化,要同时保证脐血间质干细胞的扩增能力与分化潜能,其培养和诱导分化条件的优化显得尤为重要。目的:分析筛选人脐血间质干细胞向神经元样细胞体外诱导分化过程的影响因素。设计、时间及地点:随机对照实验,于2006-08/2007-05在河南省红十字血液中心血液成分应用研究所完成。材料:脐血来自郑州市妇幼保健院正常足月分娩的胎儿,平均采血量95mL。重组人表皮生长因子、碱性成纤维细胞生长因子购自Sigma公司;分离细胞所用的四联袋为山东威高集团公司产品。方法:取采集6h内的脐血,采用四联袋密度梯度离心法分离脐血单个核细胞,以5×109L-1接种于DMEM/F12培养基中。①细胞因子实验:单独细胞因子组加入20μg/LB27、10μg/L碱性成纤维细胞生长因子;细胞因子联合组在此基础上加入5μg/L重组人表皮生长因子。②红细胞混入量实验:裂解组加入红细胞裂解液1mL,裂解后红细胞混入量为(0.44±0.13)×108/份;少红细胞组红细胞混入量为(0.51±0.21)×108/份,多红细胞组红细胞混入量为(2.65±1.28)×108/份。③首次换液时间实验:分别于接种后48h、7d首次换液。④诱导分化:将碱性成纤维细胞生长因子、重组人表皮生长因子共诱导7d的细胞爬片进行巢蛋白免疫组织化学染色。主要观察指标:观察不同因素对脐血间质干细胞增殖分化的影响。检测诱导分化后神经干细胞巢蛋白的表达。结果:培养7d后,表皮生长因子与碱性成纤维细胞生长因子联合诱导促细胞增殖的效果优于单独应用碱性成纤维细胞生长因子(t=2.880,P<0.05);红细胞裂解液组、多红细胞组贴壁细胞数明显低于少红细胞组(t=7.332~7.550,P<0.05),即混入的红细胞总量不大于108/份对细胞增殖无影响;接种后7d首次换液所获贴壁细胞数明显多于接种后48h首次换液(P<0.05)。两种细胞因子共诱导后,神经干细胞巢蛋白呈阳性表达。结论:在脐血间质干细胞向神经元样细胞诱导分化过程中,细胞因子、红细胞混入量及首次换液时间都是重要的影响因素。
BACKGROUND: Studies have shown that umbilical cord blood mesenchymal stem cells can differentiate into neuron-like cells, and at the same time to ensure the expansion and differentiation potential of umbilical cord blood mesenchymal stem cells. The optimization of culturing and inducing differentiation conditions is particularly important. OBJECTIVE: To analyze the influential factors of screening human umbilical cord blood mesenchymal stem cells to differentiate into neuron-like cells in vitro. DESIGN, TIME AND SETTING: A randomized controlled trial was performed at the Institute of Blood Composition, Henan Red Cross Blood Center from August 2006 to May 2007. Materials: Umbilical cord blood from Zhengzhou City, MCH normal fetus fetus, the average blood volume 95mL. Recombinant human epidermal growth factor, basic fibroblast growth factor were purchased from Sigma; quadruple bags used in the separation of cells for the Shandong Weigao Group products. Methods: Umbilical cord blood was harvested within 6h. Umbilical cord blood mononuclear cells were isolated by quadruple bag density gradient centrifugation and inoculated into DMEM / F12 medium at 5 × 109L-1. ① Cytokines: 20μg / LB 27, 10μg / L basic fibroblast growth factor were added into the cytokines alone group; 5μg / L recombinant human epidermal growth factor was added to the cytokines combined group. ② Erythrocyte incorporation test: 1 mL erythrocyte lysis buffer was added into lysis group, the amount of erythrocyte mixed was (0.44 ± 0.13) × 108 / l after lysis; the mixed amount of erythrocytes in less erythrocyte group was (0.51 ± 0.21) × 108 / The mixed amount of erythrocytes was (2.65 ± 1.28) × 108 / copies. ③ the first exchange of fluid experiments: respectively, 48h after inoculation, 7d first exchange fluid. ④ Induction of differentiation: Basic fibroblast growth factor and recombinant human epidermal growth factor were co-induced 7day cell cloning nestin immunohistochemical staining. MAIN OUTCOME MEASURES: The effects of different factors on the proliferation and differentiation of umbilical cord blood mesenchymal stem cells were observed. The expression of nestin in neural stem cells was detected after induction of differentiation. RESULTS: After cultured for 7 days, the effect of epidermal growth factor and basic fibroblast growth factor in inducing cell proliferation was better than that of using basic fibroblast growth factor alone (t = 2.880, P <0.05). In erythrocyte lysate group, The number of adherent cells in the erythrocyte group was significantly lower than that of the less erythrocyte group (t = 7.332-7.550, P <0.05), that is, the total amount of erythrocytes mixed was not more than 108 / part had no effect on cell proliferation; Adherent cells were significantly more than 48h after inoculation of the first exchange of fluid (P <0.05). After co-induction of both cytokines, NSCs positive for nestin. CONCLUSION: During the differentiation of umbilical cord blood mesenchymal stem cells into neuron-like cells, the amount of cytokines, the amount of erythrocytes and the time of first exchange are all important factors.