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mT/mG;Wnt1-Cre小鼠肺组织神经嵴细胞的分离培养与特征鉴定

Isolation,culture and characterization of neural crest cells from lung tissue of mT/mG;Wnt1-Cre mice
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摘要 目的体外分离培养小鼠肺组织中的神经嵴细胞(NCCs),并鉴定该细胞的特性,为研究肺损伤及修复提供新的细胞模型。方法将mT/mG双荧光报告小鼠和Wnt1-Cre转基因小鼠杂交,筛选获得增强型绿色荧光蛋白(EGFP)永久标记NCCs的mT/mG;Wnt1-Cre转基因小鼠。用酶解法制备小鼠肺组织单细胞悬液,用流式细胞仪分选获得EGFP+的细胞(即为NCCs)。用经实验室优化的DMEM/F12培养基进行原代培养,用细胞免疫荧光染色法对NCCs进行特征鉴定,使用小鼠骨髓间充质干细胞成骨诱导分化培养基定向诱导NCCs分化。结果成功通过杂交筛选得到EGFP永久标记NCCs的mT/mG;Wnt1-Cre转基因小鼠,并从肺组织中分离获得高纯度的NCCs,其能够在体外进行培养,形态为梭形,类似成纤维样贴壁增殖,NCCs表达神经嵴干细胞标志物Sox10,能被定向诱导分化为成骨细胞。结论从mT/mG;Wnt1-Cre转基因小鼠肺组织中分离、培养的NCCs增殖稳定,具有神经嵴干细胞特征,可为肺组织损伤和修复研究提供新的细胞模型。 Objective To isolate and culture neural crest cells(NCCs)from lung tissue of mice and to identify the characteristics of the cells in order to provide a new cell model for studying lung injury and injure repair.Methods The mT/mG dual-fluorescence reporter mice and Wnt1-Cre transgenic mice were hybridized,and mT/mG;Wnt1-Cre transgenic mice were screened to obtain enhanced green fluorescent protein(EGFP)permanently labeled NCCs.Cell suspension of mouse lung tissue was prepared by enzymolysis.EGFP+cells(namely NCCs)were harvested by flow cytometry.Primary culture was performed with DMEM/F12 culture medium optimized in the laboratory,NCCs was characterized by immunofluorescence microscopy.Then NCCs differentiation was directed by mouse bone marrow mesenchymal stem cells osteogenic induction.Results The mT/mG of EGFP permanently labeled NCCs was successfully obtained by hybridization and high-purity NCCs were isolated from Wnt1-Cre transgenic mice lung tissue.They can be cultured in vitro and with spindle morphology which was,similar to fibroblast adherent proliferation.NCCs expressed the neural crest stem cell marker Sox10 and induced to differentiate into osteoblasts.Conclusions NCCs isolated and cultured from lung tissue of mT/mG;Wnt1-Cre transgenic mice show stable proliferation and have the characteristics of neural crest stem cells,which may function as a potential cell model for research on lung tissue injury and the mechanism of repair.
作者 董小雯 李泳欣 龚晓雪 冯玲芳 陈俊斐 姚佳慧 楼建林 DONG Xiaowen;LI Yongxin;GONG Xiaoxue;FENG Lingfang;CHEN Junfei;YAO Jiahui;LOU Jianlin(School of Public Health,Hangzhou Medical College,Hangzhou 310013,China)
出处 《基础医学与临床》 CAS 2024年第11期1510-1515,共6页 Basic and Clinical Medicine
基金 国家重点研发计划(2019YFE0116100) 国家卫生健康委员会科学研究基金-浙江省医药卫生重大科技计划(WKJZJ-2022) 中国疾病预防控制中心化学污染与健康安全重点实验室开放基金(2024CDCKL01) 浙江省医药卫生科技计划(2024KY933)。
关键词 神经嵴细胞 原代培养 流式细胞分选 转录因子10(Sox10) neural crest cells primary culture flow cytometry SRY-box transcription factor 10(Sox10)
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