中国组织工程研究Issue(11)：1706-1711,6.DOI:10.3969/j.issn.2095-4344.2014.11.011
人胎盘微血管内皮细胞的分离培养及鉴定
Primary cultivation and identification of human placental microvascular endothelial cells
摘要
Abstract
BACKGROUND:Establishment of in vitro culture system of human placental microvascular endothelial cel s with high purity is very important. In recent studies, some scholars have successful y obtained a large number of placental microvascular endothelial cel s by three-stepenzyme digestion and magnetic separation method, but the procedures were extremely complex and it had great damage to the cel s. Therefore, how to separate human placental microvascular endothelial cel s easily and obtain high-purified cel s has become a research hotspot. <br> OBJECTIVE:To investigate an efficient method to isolate and purify human placental microvascular endothelial cel s from early vil us microvessels, observe the cel growth and identify the cel s. <br> METHODS:The vil i from normal early pregnancies (6-8 weeks) after artificial abortion were col ected aseptical y. Using two-step digestion procedure and discontinuous Percol density gradient centrifugation method, human placental microvascular endothelial cel s were obtained. Then the cel s were identified by trypsin digestion method and repeated adherence method. <br> RESULTS AND CONCLUSION:Human placental microvascular endothelial cel s were isolated successful y from early vil i. The primary cel s adhered to the wal s after inoculated for 24 hours and entered logarithmic phase at 10 days. 80%of the cel s achieved a confluence at 12-13 days after inoculating. The subculture cel s grew swiftly with the typical cobblestone appearance. Immunofluorescence staining showed that, cultured human placental microvascular endothelial cel s demonstrated a strong positive reaction to von Wil ebrand factor antigen and CD31, accounting for 100%. MTT assay results showed that, human placental microvascular endothelial cel s at passage 5 exhibited an S-shaped growth curve. High-purity human placental microvascular endothelial cel s can be obtained by proteolytic enzymes digestion and discontinuous Percol density gradient centrifugation method, and the purity is detected by trypsin digestion method and repeated adherence method.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/FⅧ因子相关抗原/CD31/MTT法/国家自然科学基金Key words
placenta/endothelial cel s/blood vessels/cel s/fluorescent antibody technique分类
医药卫生引用本文复制引用
张慧丽,陈敦金,杜培丽,方元龙,张镜,何玉甜,孙斌,肖雪,孙雯,周燕媚..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,(11):1706-1711,6.基金项目
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)@@@@the Natural Science Foundation of Guangdong Province, No. S2012010008932 （S2012010008932）
the National Natural Science Foundation of China, No.81370775,81302399 （）
Project Funds by the Education Ministry, No.20114423110004 （）
