大鼠肺血管内皮细胞的原代培养及生物学行为比较研究  

Comparative research on primary culture of pulmonary vascular endothelial cells in rats and biological behaviors

作　　者：江倩[1] 罗晓韵 刘诗韵 吴雪芬 卢文菊[1] 王健[1] 张晨婷[1] Jiang Qian;Luo Xiaoyun;Liu Shiyun;Wu Xuefen;Lu Wenju;Wang Jian;Zhang Chenting(Department of Respiratory and Critical Care Medicine,Guangzhou Institute of Respiratory Health,National Clinical Research Center for Respiratory Disease,National Center for Respiratory Medicine,State Key Laboratory of Respiratory Disease,the First Affiliated Hospital of Guangzhou Medical University,Guangzhou 510120,China;Guangzhou Regenerative Medicine and Health Guangdong Laboratory,Guangzhou 510320,China)

机构地区：[1]广州医科大学附属第一医院呼吸与危重症医学科广州呼吸健康研究院国家呼吸系统疾病临床医学研究中心国家呼吸医学中心呼吸疾病国家重点实验室,广州510120 [2]广州再生医学与健康广东省实验室,广州510320

出　　处：《国际呼吸杂志》2022年第5期381-387,共7页International Journal of Respiration

基　　金：国家自然科学基金(81800054);呼吸疾病国家重点实验室自主课题青年项目(SKLRD-QN-201904、SKLRD-QN-201922);广州市科技计划基础与应用基础研究项目(202102020019)。

摘　　要：目的建立大鼠肺动脉内皮细胞(PAEC)、肺静脉内皮细胞(PVEC)以及肺微血管内皮细胞(PMVEC)的体外分离和培养方法,分析3种肺血管内皮细胞的生物学特征及功能。方法本研究为实验研究。精分1只雄性SD大鼠肺动脉、肺静脉,肺组织取边缘剪碎,各样本分别用混合酶液消化,以血小板-内皮细胞黏附分子(PECAM-1/CD31)免疫磁珠法分选内皮细胞,用内皮细胞专用培养基培养。倒置显微镜下观察3种肺血管内皮细胞的形态差别,通过免疫荧光鉴定各组细胞纯度。通过免疫荧光和Western blot观察3组细胞血管性血友病因子(vWF)和平滑肌激动蛋白抗体(α-SMA)蛋白表达情况。通过基质胶体外成环实验比较3组细胞1.5、2.5、3.5、4.5、5.5、6.5、7.5 h成环情况。采用SPSS 22.0和FlowJo 10软件进行统计学分析和统计作图。结果3组细胞24 h后均迅速贴壁,vWF免疫荧光染色均为阳性。3组细胞形态均为铺路石状,其中PAEC为典型的铺路石状,大多数呈椭圆形;而PVEC、PMVEC形态更细长。免疫荧光显示PVEC和PMVEC细胞浆内少量α-SMA蛋白弥散表达[(0.16±0.03)、(0.23±0.01)],而PAEC中几乎观察不到α-SMA蛋白表达(0.06±0.01)。随着时间推移,3种肺血管内皮细胞成环周长均呈先上升后下降趋势,其中PVEC组成环长度最长。3组细胞成环周长在组间、时点间、时间和组间交互作用差异均具有统计学意义(P值均<0.01)。结论该方法所获得的3种肺血管内皮细胞纯度高,可在体外稳定培养。3种血管内皮细胞生物学功能有差别,其中PVEC体外成环能力最强。PAEC细胞表达α-SMA蛋白量最少。Objective To study the biological characteristics and functions of three types of pulmonary vascular endothelial cells by setting up the methods for isolation and culture of pulmonary artery endothelial cells(PAEC),pulmonary vein endothelial cells(PVEC)and pulmonary microvascular endothelial cells(PMVEC)in vitro.Methods This is an experimental study.The limbic of pulmonary arteries,pulmonary veins and pulmonary tissues from one male rat were finely divided and cut into pieces,types of samples were digested with mixed enzyme solutions,the endothelial cells were sorted out with magnetic activated cell sorting for platelet endothelial cell adhesion molecules(PECAM-1/CD31),and cultured with special medium for endothelial cells.The morphological differences of cells from different tissues under an inverted microscope were observed,and the purity of cells was identified with immunofluorescence staining.The expressions of von Willebrand factor(vWF)andα-smooth muscle actin antibody(α-SMA)proteins in groups of cells were observed by immunofluorescence and Western blot;the looping details of cells in groups were compared by Matrigel in vitro looping experiments lasting 1.5,2.5,3.5,4.5,5.5,6.5 and 7.5 hours.SPSS 22.0 and FlowJo 10 software were applied for statistical analysis and statistical mapping.Results The endothelial cells were quickly adherent in three groups after 24 hours,and the immunofluorescence staining of vWF was positive.The morphological characteristics of endothelial cells exhibited paving-stone patterns,PAEC showed typical paving-stone patterns and oval morphologies,PVEC and PMVEC showed significantly thinner and longer morphologies.The immunofluorescence staining showedα-SMA in the cytoplasm of PVEC and PMVEC was dispersively expressed([0.16±0.03],[0.23±0.01]);while the expression ofα-SMA protein(0.06±0.01)in PAEC was barely observed.The circumferential length of three kinds of pulmonary vascular endothelial cells increased firstly and then decreased over time,and the looping length of PVEC was th

关 键 词：肺动脉 肺静脉 微血管 内皮细胞 细胞培养技术 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]