改良人视网膜微血管内皮细胞原代培养及鉴定  

Primary culture and characterization of mi-crovascular endothelial cells from human retina by modified method

作　　者：刘洪雷[1] 惠延年[1] 刘军[2] 王雨生[1] 

机构地区：[1]第四军医大学西京医院眼科,陕西省西安市710032 [2]第四军医大学唐都医院心脏内科,陕西省西安市710038

出　　处：《眼科新进展》2005年第3期197-200,共4页Recent Advances in Ophthalmology

基　　金：国家自然科学基金资助(编号:30371516)~~

摘　　要：目的建立人视网膜微血管内皮细胞的改良培养方法并研究其体外免疫组化特征。方法由人微血管片断生长出的内皮细胞首先通过CD31+免疫磁珠标记,于明胶包被培养瓶接种,采用含有20mg·L-1内皮细胞生长因子、90mg·L-1肝素、150mL·L-1马血清和100mL·L-1人血清的内皮细胞基本培养基;非内皮细胞例如周细胞可机械除杂,保留的内皮细胞克隆特征性的标志物为CD31+磁珠标记;特征通过免疫组化和激光共聚焦显微镜评价。人视网膜微血管内皮细胞的生长曲线通过MTT法测定。结果在这样的培养条件下,内皮细胞1-2d由微血管片断游出,1周可形成较大克隆,大约2周可融合成单层细胞。这些细胞证实可被CD31+磁珠附着,表达von Willebrand因子、细胞粘附蛋白CD31与CD34,并不表达α-平滑肌肌动蛋白。内皮细胞可部分混杂其他细胞,持续生长并传代。结论本研究为采用磁珠鉴定人源性视网膜微血管内皮细胞的培养及鉴定的新改良方法。本培养实验为研究人眼异常血管疾病提供了新的平台。Objective To develop modified methods for the culture of mi-crovascular endothelial cells (hMVECs) from human retina and to investigate their propagation and survival in vitro. Methods Endothelial cells from human capillary fragments were first identified with CD31 coupled magnetic beads and cultured on gelatin-coated dishes in endothelial basic medium (EBM) containing 20 mg·L-1 endothelial growth factor,90 mg·L-1 heparin,15% horse serum and 10% human serum. Non-EC, such as pericytes, were removed manually. Endothelial cell-specific properties were the cell clone with CD31 coupled magnetic beads and assessed by immunohistochemical staining and laser confocal scanning microscope. The growth curve of hMVECs were assessed by MTT assay. Results Under these culture conditions,migrating cells emerged from capillary fragments after 1 to 2 days and formed large clonies by 1 week, formed a confluent monolayer by 2 weeks. These cells demonstrated attached with CD31 coupled with magnetic beads,expressed von Willebrand factor and the cell adhesion protein CD31 and CD34, and did not contain smooth muscle α-actin; and the clone might be mixed with Non-EC partially; the hMVECs could be maintained and passaged in vitro. Conclusion This study is the new description of culture and characterization of purified retinal ECs from human retina selected by magnetic beads. These cultures will be highly relevant to the studies of abnormal vascular disease in human eye.

关 键 词：内皮细胞 细胞培养 CD31磁珠 CD34 免疫组化 增生 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]