摘要
为了建立一种自主分离原代犬血管内皮细胞(vein endothelial cell,VEC)的新方法,试验将无菌分离的犬降主动脉由内向外翻转后,放入0.075%的Ⅰ型胶原酶中消化15 min,然后轻刮血管内表面,加入0.1%的Ⅰ型胶原酶消化4 min;经内皮细胞培养基(endothelial cell medium,ECM)重悬细胞并贴壁培养24 h后,对特征细胞进行准确标记,刮除其他干扰细胞,快速消化特征细胞集落并扩大培养,得到原代细胞;对原代细胞进行形态观察、生长特性分析,以及细胞表面标志物鉴定(免疫荧光法和PCR法)和细胞纯度分析。结果表明:原代细胞培养24 h后,在倒置显微镜下可见散在的、成群分布的“鹅卵石”状或“铺路石”状细胞。不同代次细胞生长曲线均呈典型的“S”型;P3细胞(传代至第3代的细胞)、P6细胞(传代至第6代的细胞)的增殖活性比P8细胞(传代至第8代的细胞)强。分离纯化后的细胞经免疫荧光法及PCR法鉴定发现,内皮细胞表面标志物——血小板-内皮细胞黏附分子(CD31)、造血祖细胞抗原CD34、血管性血友病因子(vWF)、血管内皮生长因子(VEGF)和血管内皮钙黏蛋白(VE-Cadherin)呈阳性表达;其中,阳性表达CD31的细胞占比为(94.22±1.87)%,阳性表达造血祖细胞抗原CD34的细胞占比为(96.24±1.09)%。说明本试验通过准确标记细胞、清除干扰细胞并快速消化所需细胞,得到1株纯度高且具备稳定生长特性的原代犬VEC系,操作切实有效,可重复性高,为VEC的高效分离与培养提供了一种简便的方法。
In order to establish a new method for autonomous isolation of primary canine vascular endothelial cells(VEC),in the experiment,after the sterile separated canine descending aorta was turned from inside to outside,it was put into 0.075%type I collagenase for digestion for 15 minutes;then the inner surface of the blood vessel was gently scraped and 0.1%type I collagenase was added to digest for 4 min.After resuspension of cells in endothelial cell medium(ECM)and adherent culture for 24 h,the characteristic cells were accurately labeled and other interfering cells were scraped.After rapid digestion of characteristic cell colonies and expansion of culture,primary cells were obtained.Analysis of morphological observation and growth characteristics was carried out in primary cells,as well as identification of cell surface markers(immunofluorescence method and PCR method)and cell purity analysis.The results showed that when primary cells were cultured for 24 h,scattered,clustered“pebble”or“paving stone”cells were visible under an inverted microscope.The growth curves of different generations of cells were typical“s”shapes;P3 cells(cells passaged to passage 3)and P6 cells(cells passaged to passage 6)had stronger proliferative activity than P8 cells(cells passaged to passage 8).After isolation and purification of cells,immunofluorescence method and PCR identification method showed that endothelial cell surface markers:platelet endothelial cell adhesion molecule-1(CD31),hematopoietic progenitor cell antigen CD34,von willebrand factor(von willebrand factor[vWF],vascular endothelial growth factor[VEGF]and vascular endothelial cadherin[VE-Cadherin ])were positively expressed.Among them,the proportion of cells that actively expressed CD31 was(94.22±1.87)%,and the proportion of cells that actively expressed CD34 was(96.24±1.09)%.The results suggested that by accurately labeling cells,removing interfering cells and rapidly digesting the required cells in this experiment,one primary canine VEC line with high purity and stable growth characteristics was obtained,which was effective and highly reproducible,and provided a convenient method for efficient isolationandcultureof VEC.
作者
赖健仪
冼伟杭
陈沃俊
邓翔云
张妙齐
曾霭宁
刘璨颖
陈胜锋
陈志胜
白银山
王丙云
LAI Jianyi;XIAN Weihang;CHEN Wojun;DENG Xiangyun;ZHANG Miaoqi;ZENG Aining;LIU Canying;CHEN Shengfeng;CHEN Zhisheng;BAI Yinshan;WANG Bingyun(School of Life Science and Engineering,Foshan University,Foshan 528000,China)
出处
《黑龙江畜牧兽医》
北大核心
2023年第16期92-97,138,共7页
Heilongjiang Animal Science And veterinary Medicine
基金
广东省自然科学基金项目(2020A1515011110)
广东省普通高校动物干细胞工程技术研究中心项目(2021GCZX006)。
关键词
犬
降主动脉
血管内皮细胞
细胞培养
细胞纯化
细胞鉴定
dogs
descending aorta
vascular endothelial cells
cell culture
cell purification
cell identification
