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人腺样体间充质干细胞向嗅感觉神经元诱导分化的体外研究

Study on in vitro differentiation of human adenoid-derived mesenchymal stem cells into olfactory sensory neurons
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摘要 目的探讨体外分离和培养人腺样体组织来源的间充质干细胞(adenoid-derived mesenchymal stem cells,aMSCs)的可行性,并诱导观察aMSCs向嗅感觉神经元的分化。方法收集2020年9—11月来源于中南大学湘雅二医院腺样体肥大患儿的手术切除腺样体组织,胰蛋白酶消化联合贴壁法培养P0细胞,传代培养,流式细胞技术检测P5代细胞表面抗原CD45、CD73、CD90的表达,观察成骨、成脂诱导分化能力。采用维甲酸(retinoic acid,RA)、音猬因子(sonic hedgehog,SHH)、碱性成纤维生长因子(basic fibroblast growth factor,bFGF),以及RA+SHH、RA+bFGF、SHH+bFGF和RA+SHH+bFGF分别诱导分化P5代aMSCs,倒置显微镜下观察细胞形态,免疫荧光抗体法检测细胞表面感觉神经元标志物β-tubulin 3、嗅感觉神经元标志物生长相关蛋白-43(growth associated protein-43,GAP43)和嗅标记蛋白(olfactory maker protein,OMP)的表达。采用两个样本率的四格表资料的χ2检验比较表达强度的差异。结果P0代aMSCs具有良好的贴壁和增殖性能,P2代细胞已基本纯化。P5代细胞阳性表达CD73和CD90,其纯度分别为99.3%和97.75%,不表达CD45,成骨和成脂诱导分化良好。RA、SHH、bFGF诱导分化细胞均成神经元样外观,β-tubulin 3阳性表达;bFGF+SHH组和RA+SHH+bFGF组诱导细胞均表达GAP43,后组表达阳性更强(χ^(2)=17.48,P<0.005);所有诱导组均未见OMP阳性表达。结论腺样体组织可培养获得能稳定传代、具有良好分化能力的aMSCs。aMSCs是间充质干细胞家族新成员,具有向神经元分化的能力,RA、SHH和bFGF体外联合诱导能促使其分化为未成熟嗅感觉神经元。 Objective To investigate the feasibility of isolation and culture of human adenoid-derived mesenchymal stem cells(aMSCs)in vitro,and to observe the differentiation of aMSCs into olfactory sensory neurons.Methods Adenoid tissues surgically removed from children with adenoid hypertrophy in the Second Xiangya Hospital of Central South University from September to November of 2020 were collected.The adenoid tissues were digested and isolated by trypsin and then cultured with adhesion method.The expressions of cell surface antigens CD45,CD73 and CD90 on aMSCs of P5 generation were tested by flow cytometry,and the ability of osteogenic and adipogenic induction were used to identify cell differentiation ability.Then,aMSCs were induced into differentiation by retinoic acid(RA),sonic hedgehog(SHH),basic fibroblast growth factor(bFGF),RA+SHH,RA+bFGF,SHH+bFGF and RA+SHH+bFGF,respectively.The morphology of differentiated cells was observed under inverted microscope.The expression ofβ-tubulin 3,which was the specific marker of sensory neuron,the expressions of growth associated protein-43(GAP43)and olfactory maker protein(OMP),which were the specific markers of olfactory sensory neuron,were detected by immunofluorescence antibody assay.The expression intensities were compared by Chi-square test of four-grid table data.Results aMSCs were successively isolated and cultured from human adenoid tissues.P0 cells generation had good adhesion and proliferation performance.P2 cells were basically purified.P5 cells expressed CD73 and CD90 with the purity of 99.3%and 99.75%respectively,without CD45 expression.P5 cells had a good ability of osteogenic differentiation and adipogenic differentiation.Neuron-like morphology and expression ofβ-tubulin 3 were found in differentiated cells after induced by RA,SHH,or bFGF,respectively.An induction of expression of GAP43 was found in differentiated cells of bFGF+SHH group and RA+SHH+bFGF group,without expression of OMP of each group.The intensity of GAP43 expression of RA+SHH+bFGF group was stronger than that of bFGF+SHH group(χ^(2)=17.48,P<0.005).Conclusions aMSCs can be cultured from human adenoid tissues,with the stably passaged and good differentiation ability.As a new population of mesenchymal stem cells,aMSCs have the neuroregenerative properties and could differentiate into immature olfactory sensory neurons under the induction of RA+SHH+bFGF in vitro.
作者 郭芳芳 余本铨 陈勇 何郡 谷钰 万鑫 肖自安 Fangfang Guo;Benquan Yu;Yong Chen;Jun He;Yu Gu;Xin Wan;Zian Xiao(Department of Otorhinolaryngology Head and Neck Surgery,Second Xiangya Hospital of Central South University,Changsha 410011,China;National Health Commission(NHC)Key Laboratory of Birth Defect for Research and Prevention,Hunan Provincial Maternal and Child Health Care Hospital,Changsha 410008,China)
出处 《中华耳鼻咽喉头颈外科杂志》 CSCD 北大核心 2023年第3期233-239,共7页 Chinese Journal of Otorhinolaryngology Head and Neck Surgery
基金 中南大学研究生科研创新项目(2021zzts1071)。
关键词 腺样体 间充质干细胞 体外诱导 嗅感觉神经元 Adenoid Mesenchymal stem cell Induction in vitro Olfactory sensory neuron
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