大鼠主动脉血管内皮细胞组织块天花板培养法提取及鉴定  

Isolation and identification of rat aortic endothelial cells using the tissue explant ceiling culture method

作　　者：张帆[1] 李柏霖 池茗 刘海琴 唐元瑜[2] Zhang Fan;Li Bolin;Chi Ming;Liu Haiqin;Tang Yuanyu(College of Acupuncture and Massage,Fujian University of TCM,Fuzhou 350122,China;College of TCM,Fujian University of TCM,Fuzhou 350122,China;College of Integrated Traditional Chinese and Western Medicine,Fujian University of TCM,Fuzhou 350122,China;Long March Hospital Affiliated to Naval Medical University,Shanghai 200003,China)

机构地区：[1]福建中医药大学针灸推拿学院,福州350122 [2]福建中医药大学中医学院,福州350122 [3]福建中医药大学中西医结合学院,福州350122 [4]海军军医大学第二附属医院上海长征医院,上海200003

出　　处：《中国组织化学与细胞化学杂志》2025年第1期61-65,85,共6页Chinese Journal of Histochemistry and Cytochemistry

基　　金：国家自然科学基金资助课题(81072714);福建省科技厅自然科学基金资助课题(2017J01545)。

摘　　要：目的运用组织块天花板培养法体外分离大鼠主动脉血管内皮细胞(aortic vascular endothelial cells,AECs),为进一步开展高血压动脉粥样硬化、肺动脉高压等血管性疾病研究提供重要的工具细胞。方法取60 g SD大鼠1只,无菌分离出胸、腹主动脉,剔除血管外筋膜、脂肪组织后,将内膜外翻,予0.1%Ⅱ型胶原酶预消化15 min后,将其剪碎成米粒状大小,采用“组织块天花板法”进行原代培养。通过细胞形态学观察、免疫细胞化学染色法检测第Ⅷ因子相关抗原及血管形成实验鉴定所培养的目的细胞。结果培养2 d后,少量短梭形细胞从血管组织块边缘爬出;4 d后“岛屿状”的细胞集落形成;6~8 d后集落融合成片,呈典型的单层、“鹅卵石样”、镶嵌式排列;第Ⅷ因子相关抗原免疫细胞化学染色显示胞质淡染成棕红色,阳性细胞率达99%以上;血管形成实验为阳性。结论“组织块天花板培养法”能够简单、高效地提取原代大鼠AECs。Objective To establish a tissue explant ceiling culture method for isolating rat aortic vascular endothelial cells(AECs)as an important tool for further research on vascular diseases such as hypertension,atherosclerosis,and pulmonary arterial hypertension.Methods A 60 g SD rat was used to aseptically isolate the thoracic and abdominal aorta.After removing the vascular adventitia and adipose tissue,the intima was everted and pre-digested with 0.1%type II collagenase for 15 min.The aorta was then cut into pieces approximately the size of rice grains,and primary culture was conducted using the“tissue explant ceiling culture method”.The target cells were identified through morphological observation,immunocytochemical staining for von Willebrand factor(vWF)-related antigen,and tube formation assays.Results After 2 days of culture,a small number of spindle-shaped cells began to migrate out from the edges of the vascular explants.By day 4,“island-like”cell colonies had formed,and by days 6 to 8,these colonies had fused into a monolayer with a characteristic“cobblestone-like”mosaic arrangement.Immunocytochemical staining for vWF-related antigen showed light brown-red cytoplasmic staining,with a positive cell rate exceeding 99%.The tube formation assay was positive.Conclusion The“tissue explant ceiling culture method”provides a simple and efficient approach for isolating primary rat AECs.

关 键 词：大鼠 主动脉血管内皮细胞 组织块天花板培养法 第Ⅷ因子相关抗原 血管形成实验 

分 类 号：R329.21[医药卫生—人体解剖和组织胚胎学]