原代大鼠主动脉血管干细胞的体外培养扩增及鉴定  

Expansion and identification of primary rat aortic vascular stem cells in vitro

作　　者：马华根 黄燕 杨盈欣 刘海琴 唐元瑜[4] 丛伟红[1] MA Huagen;HUANG Yan;YANG Yingxin;LIU Haiqin;TANG Yuanyu;CONG Weihong(Xiyuan Hospital,China Academy of Chinese Medical Sciences,Beijing 100091,China;School of Traditional Chinese Medicine,Beijing University of Chinese Medicine,Beijing 100029,China;Shanghai Changzheng Hospital,Naval Medical University,Shanghai 200003,China;School of Traditional Chinese Medicine,Fujian University of Traditional Chinese Medicine,Fuzhou 350122,China)

机构地区：[1]中国中医科学院西苑医院,北京100091 [2]北京中医药大学中医学院,北京100029 [3]海军军医大学上海长征医院,上海200003 [4]福建中医药大学中医学院,福建福州350122

出　　处：《南方医科大学学报》2024年第10期1881-1886,共6页Journal of Southern Medical University

基　　金：国家自然科学基金(81973594)。

摘　　要：目的采用“切段外膜贴壁培养法”体外培养扩增出原代大鼠主动脉血管干细胞,为开展血管重构及相关疾病研究提供实用的工具细胞。方法无菌分离2~3周龄Sprague-Dawley大鼠胸腹主动脉,切成长约2.0 mm的血管节段,均匀种瓶,经外膜贴壁固化后进行原代培养,待细胞生长融合度达80%~90%时传代。镜下观察细胞形态及其生长特性,采用流式细胞仪分析目的细胞表面标志物CD分子表达情况,成脂、成骨诱导分化实验检测其多向分化潜能。结果接种3 d后,少量梭形、星形或多角形的细胞从血管节段周围爬出;5~6 d后形成岛屿状细胞集落,细胞增殖迅速,呈放射状向外扩大,并出现细胞克隆现象;7~8 d后细胞融合成片,呈涡旋状分布;传至第3代后,细胞匀质性较高,表现为典型的“成纤维样”排列生长。流式细胞仪分析细胞主要表达CD44、CD73、CD90,阳性率分别为80.3%、62.2%、46.8%;低表达CD34、CD45、CD11b/c,阳性率分别为1.1%、0.2%、0.2%。体外诱导分化实验表明目的细胞具有成脂、成骨分化潜能。结论“切段外膜贴壁培养法”可成功分离培养出具有间充质干细胞特性的大鼠主动脉血管干细胞。Objective To culture and expand primary rat aortic vascular stem cells in vitro and evaluate their characteristics as mesenchymal stem cells.Methods The thoracic and abdominal aortas isolated from 2-to 3-week-old Sprague-Dawley rats were cut into vascular segments 2.0 mm in length and cultured in culture flasks till adhesion and solidification of the outer membranes.The primary cells were further cultured to 80%-90%confluence before passaging.The morphology and growth characteristics of the cells were observed under a microscope,and the expressions of surface marker CD molecules on the cells was analyzed using flow cytometry.Adipogenic and osteogenic differentiation assays were performed to assess the capacity of the cells for multilineage differentiation.Results After 3 days of culture,a small number of spindle,star-shaped or polygonal cells migrated out from the peripheral of the vascular segments.At 5-6 days,island-like cell clusters occurred and the cells began to proliferate rapidly.The cell clusters expanded radially and showed signs of cell cloning.At 7-8 days,the cells fused into sheets and displayed a vortex-like distribution.The cells in the third passage presented with a uniform morphology,showing a typical fibroblast-like arrangement.Flow cytometry showed that the cells expressed predominantly CD44(80.3%),CD73(62.2%)and CD90(46.8%)with low expressions of CD34(1.1%),CD45(0.2%)and CD11b/c(0.2%).Adipogenic and osteogenic differentiation experiments demonstrated that the cells were capable of lipogenic and osteogenic differentiation in vitro.Conclusion Rat aortic vascular stem cells with mesenchymal stem cell characteristics can be successfully isolated and cultured by adherent culture of the segmented outer membrane.

关 键 词：血管干细胞 主动脉 切段外膜贴壁培养法 原代培养 免疫表型 分化潜能 血管重构 

分 类 号：R318[医药卫生—生物医学工程]