人主动脉内皮细胞体外培养方法的建立及生物学活性鉴定  

Establishment of human aortic endothelial cell culture in vitro and identification of biological activity

作　　者：何珂帅 章辉[2] 张俊杰 吴亚 武晓泓[3] 邵永丰 He Keshuai;Zhang Hui;Zhang Junjie;Wu Ya;Wu Xiaohong;Shao Yongfeng(Department of Cardiovascular Surgery;Public Experiment Platform;Department of Endocrinology, the First Affiliated Hospital of NMU , Nanjing 210029, China)

机构地区：[1]南京医科大学第一附属医院心脏大血管外科,江苏南京210029 [2]南京医科大学第一附属医院公共平台实验室,江苏南京210029 [3]南京医科大学第一附属医院内分泌科,江苏南京210029

出　　处：《南京医科大学学报（自然科学版）》2018年第6期734-738,共5页Journal of Nanjing Medical University(Natural Sciences)

基　　金：江苏省自然科学基金(BK20151590)

摘　　要：目的:建立体外人主动脉内皮细胞(human aortic endothelial cells,HAECs)提取和培养的方法。方法:从术中取下的人主动脉组织上剥离内皮层,Ⅰ型胶原酶消化分离HAECs,利用显微镜观察其生长状况,免疫组织化学方法及流式细胞法鉴定及检测细胞纯度,以氧化低密度脂蛋白(oxidized low-density lipoprotein,ox-LDL)检测细胞吞噬功能并检测细胞在体外的血管生成能力。结果:0.1%及0.2%的Ⅰ型胶原酶,最佳消化时间均为50 min,2 h贴壁细胞数分别(25.2±2.1)×103,(40.5±3.8)×103)个。细胞融合后在倒置显微镜下呈"鹅卵石"样排列生长。血小板-内皮黏附分子(platelet endothelial cell adhesion molecule-1,CD31)及Ⅷ因子鉴定为阳性,细胞纯度达(96.43±4.12)%。细胞内吞ox-LDL及血管生成能力较好。结论:钝性分离主动脉内皮层,辅以Ⅰ型胶原酶消化是一种较好的HAECs获取方法,酶浓度及消化时间是其重要的影响因素。Objective：To Establish the methods of extracting and cultivate human aortic endothelial cells（HAECs）in vitro.Methods：Stripping endodermis from human aorta,using collagenaseⅠto digest endothelial cells from endodermis,and then utilizing immunohistochemistry and flow cytometry（FCM）to detect purity of HAECs. Results：0.1% and 0.2% collagenase type Ⅰ,the best digestion time was 50 min,the adherent cells number in 2 h were（25.2 ± 2.1）×103,（40.5 ± 3.8）×103. After cell fusion,cells were cobblestone-like growth in inverted microscope. Cells were identified by CD31 and Ⅷ factors as positive,the cell purity reached（96.43 ± 4.12）%. The ability of endocytosis of ox-LDL and angiogenesis were normal. Conclusion：The method that blunt dissection of the aortic endothelium and simply use type Ⅰ collagenase digestion was better to separate HAECs. Enzyme concentration and digestion time are important factors. The method is simple and convenient,shortening the training cycle and obtaining high purity cells.It is beneficial to the endothelial cells experiment in vitro.

关 键 词：人主动脉内皮细胞 免疫组织化学 细胞分离 细胞培养 流式细胞术 

分 类 号：R329.26[医药卫生—人体解剖和组织胚胎学]