人胎盘微血管内皮细胞的分离培养及鉴定

人胎盘微血管内皮细胞的分离培养及鉴定 1，2122111111张慧丽，杜培丽，方元龙，张 镜，何玉甜，孙 斌，肖 雪，孙 雯，周燕媚，陈敦金 12广州医科大学附属第三医院妇产科，广州市重症孕产妇救治中心，广州市妇产科研究所，广东省广州市 510150;广州医科 大学，广东省广州市 510000 Primary cultivation and identification of human placental microvascular endothelial cells 1, 2122111Zhang Hui-li, Du Pei-li, Fang Yuan-long, Zhang Jing, He Yu-tian, Sun Bin, Xiao Xue, 111Sun Wen, Zhou Yan-mei, Chen Dun-jin 1Department of Gynecology and Obstetrics, the Third Affiliated Hospital of Guangzhou Medical University, Obstetric Critical Care Center of Guangzhou, Guangzhou Institute of Gynecology and Obstetrics, Guangzhou 2510150, Guangdong Province, China; Guangzhou Medical University, Guangzhou 510000, Guangdong Province, China 背景:建立高纯度的人胎盘微血管内皮细胞体外培养体系对研究胎盘功能是十分必要的。目前，国内外研究多采用三 步酶消化法结合免疫磁珠法分离胎盘微血管内皮细胞，然而步骤过于繁琐且对细胞损伤较大。因而，如何简化人胎盘 微血管内皮细胞分离步骤，同时又能提高目标细胞纯度的体外培养成为研究热点。 目的:探索一种简便高效的从早期绒毛组织中分离人胎盘微血管内皮细胞的体外培养方法，观察细胞生长状态并进行 鉴定。 方法:利用健康孕6-8周孕妇行人工流产后的绒毛组织，经两步酶消化法和非连续 Percoll密度梯度离心得到人胎盘 微血管内皮细胞，传代时利用胰酶消化法和反复贴壁法对细胞进行纯化。 结果与结论:实验成功获取人胎盘微血管内皮细胞，原代培养的人胎盘微血管内皮细胞在培养24 h后基本完全贴壁， 第10天进入对数生长期，第12至13天细胞浓度达80%，传代细胞较原代细胞生长活跃，培养5-7 d可长满培养 瓶底，呈“铺路石样”排布。免疫荧光化学结果显示，培养的细胞中F?因子与CD31相关抗原双荧光染色呈阳性，细 胞阳性率达100%。MTT法测得培养第5代人胎盘微血管内皮细胞的生长曲线呈倒“S”形。结果证实，应用两步酶消 化法、非连续 Percoll密度梯度离心法分离细胞，并利用胰酶消化法和反复贴壁法纯化细胞，可获得大量高纯度的人 胎盘微血管内皮细胞。 中国组织研究杂志出版内容重点:组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质 疏松;组织工程 全文链接: 关键词 : 组织工程; 血管内皮细胞; 人胎盘微血管内皮细胞; 早期绒毛组织; 原代培养; 免疫细胞荧光化学; 细胞鉴 定; 胰酶消化; 纯化; F?因子相关抗原;CD31; MTT法; 国家自然科学基金; Abstract: BACKGROUND: Establishment of in vitro culture system of human placental microvascular endothelial cells with high purity is very important. In recent studies, some scholars have successfully obtained a large number of placental microvascular endothelial cells by three-stepenzyme digestion and magnetic separation method, but the procedures were extremely complex and it had great damage to the cells. Therefore, how to separate human placental microvascular endothelial cells easily and obtain high-purified cells has become a research hotspot. OBJECTIVE: To investigate an efficient method to isolate and purify human placental microvascular endothelial cells from early villus microvessels, observe the cell growth and identify the cells. METHODS: The villi from normal early pregnancies (6-8 weeks) after artificial abortion were collected aseptically. Using two-step digestion procedure and discontinuous Percoll density gradient centrifugation method, human placental microvascular endothelial cells were obtained. Then the cells were identified by trypsin digestion method and repeated adherence method. RESULTS AND CONCLUSION: Human placental microvascular endothelial cells were isolated successfully from early villi. The primary cells adhered to the walls after inoculated for 24 hours and entered logarithmic phase at 10 days. 80% of the cells achieved a confluence at 12-13 days after inoculating. The subculture cells grew swiftly with the typical cobblestone appearance. Immunofluorescence staining showed that, cultured human placental microvascular endothelial cells demonstrated a strong positive reaction to von Willebrand factor antigen and CD31, accounting for 100%. MTT assay results showed that, human placental microvascular endothelial cells at passage 5 exhibited an S-shaped growth curve. High-purity human placental microvascular endothelial cells can be obtained by proteolytic enzymes digestion and discontinuous Percoll density gradient centrifugation method, and the purity is detected by trypsin digestion method and repeated adherence method. 中国组织工程研究杂志出版内容重点:组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质 疏松;组织工程 全文链接: Key words: placenta; endothelial cells; blood vessels; cells; fluorescent antibody technique; 中图分类号: R394.2 基金资助: 广东省自然科学基金(S2012010008932);国家自然科学基金(81370775，81302399);教育部项目 基金(20114423110004) 通讯作者: 陈敦金，主任医师。广州医科大学附属第三医院妇产科，广州市重症孕产妇救治中心，广州市妇产科研究 所，广东省广州市 510000 作者简介: 张慧丽，女，1987年生，山东省烟台市人，汉族，广州医科大学在读硕士。 引用本文: 张慧丽,杜培丽,方元龙等. 人胎盘微血管内皮细胞的分离培养及鉴定[J]. 中国组织工程研究, 2014, 18(11): 1706-1711.