人视网膜微血管周细胞原代培养及鉴定  

Primary culture and characterization of microvascular pericytes from human retina

作　　者：刘洪雷[1] 王雨生[1] 刘军[2] 惠延年[1] 

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

出　　处：《国际眼科杂志》2005年第1期55-58,共4页International Eye Science

摘　　要：目的:建立人视网膜微血管周细胞的培养方法并研究体外其免疫组化特征。方法:由人微血管片断生长出的周细胞首先通过其形态及生长方式进行鉴别,采用含有200mL/L胎牛血清的DMEM培养基于未包被培养瓶培养;非周细胞例如色素上皮细胞可机械除杂;周细胞特征性的标志物通过免疫组化和激光共聚焦显微镜评价。人视网膜微血管周细胞的生长曲线通过MTT法测定。结果:在这样的培养条件下,周细胞1～2d由微血管片断游出,1wk可形成较大克隆,大约2wk可融合成单层细胞。这些细胞为高度不规则形,重叠生长方式,没有接触抑制;表达α-平滑肌肌动蛋白,肌动蛋白以及3G5阳性;不含有von Willebrand因子和角蛋白;周细胞可部分混杂其他细胞,纯度为99%;周细胞可持续生长并传代。结论:本研究为首次报道人源性视网膜微血管周细胞的培养及鉴定。本培养实验为研究人眼异常血管疾病提供了新的平台。AIM: To develop methods for the culture of microvascular pericytes (PC) from human retina and to investigate their immunocytochemical characteristics in vitro .· METHODS: Pericytes from human retinal capillary fragments were first identified from morphology and patterning of growth and then cultured on non-coated dishes in Dulbecco's modified Eagle's medium(DMEM) containing 20% fetal bovine serum. Non-PCs, such as retinal pigment epithelial cells, were removed manually. Pericyte-specific markers were assessed by immunocytochemical staining and laser scanning confocal microscope. The growth curve of hPC were assessed by MTT assay.· RESULTS: Under these culture conditions, migrating cells emerged from retinal capillary fragments after 1 to 2d and formed large clonies at 1wk and a confluent monolayer at about 2wk. These cells demonstrated high irregular cell and overlapping growth pattern, without contact inhabit. α-smooth muscle actin (SMA) and actin expressed. They reacted with monoclone antibody 3G5, but did not contain von Willebrand factor or keratin. The pericytes may be mixed with Non-hPC partially with the purity ratio of 99%. The hPC could be subcultured and cultivated for numerous population doubling in vitro .· CONCLUSION: This study is the first description of culture and characterization of purified retinal PC from human retina. These cultures provide a new tool for the research of abnormal vascular disease in human eyes.·

关 键 词：周细胞 微血管 视网膜 免疫组化 原代培养 生长方式 Α-平滑肌肌动蛋白 培养方法 结论 特征 

分 类 号：R774[医药卫生—眼科] R735.2[医药卫生—临床医学]