人卵巢上皮癌微血管内皮细胞分离培养与鉴定  

Primary culture and identification of human epithelial ovarian cancer microvascular endothelial cell

作　　者：刘婷[1,2] 杜雪莲[1] 盛修贵[1] 颜春晓[3] 王学政[2,4] 蔡维望[2,4] 

机构地区：[1]山东省肿瘤医院妇瘤科,山东济南250117 [2]济南大学.山东省医学科学院医学与生命科学学院,山东济南250000 [3]山东省医药生物技术研究中心,山东济南250000 [4]临沂市肿瘤医院妇瘤科,山东临沂276000

出　　处：《中华肿瘤防治杂志》2013年第15期1168-1171,共4页Chinese Journal of Cancer Prevention and Treatment

基　　金：国家自然科学基金(30901713);山东省自然科学基金(BS2010YY065)

摘　　要：目的:建立人卵巢上皮癌微血管内皮细胞的原代分离纯化及培养方法。方法:收集2012-03-01-2012-10-31山东省肿瘤医院妇瘤科手术切除的卵巢上皮癌标本21例,采用胶原酶消化法分离人卵巢上皮癌微血管内皮细胞,免疫磁珠法提取纯化内皮细胞。采用形态学观察,免疫荧光技术及自发管腔形成实验对提取的内皮细胞进行鉴定;流式细胞仪检测细胞纯度;MTT法测定细胞增殖并绘制细胞生长曲线。结果:提取人卵巢微血管内皮细胞呈圆形或类圆形,周围有抗CD31免疫磁珠附着,活细胞计数测定>95%,流式细胞仪检测纯度>95%,细胞贴壁后呈长梭形、星形或多角形,胞内含有CD31磁珠,3～6d细胞间互相融合呈单层片状生长,铺满后呈典型铺路石样特征,荧光显微镜下观察表达内皮细胞特异性标志vWF,管腔形成实验证实体外有二维管腔形成。通过观察发现,内皮细胞24h内贴壁,7～11d可完全融合并出现接触抑制现象。通过MTT法测定内皮细胞生长分为增殖期、对数增长期和平台期3个阶段,生长曲线为"S"型。结论:应用免疫磁珠法成功建立了一种操作简单,且可获得纯度高和活性好的人卵巢上皮癌微血管内皮细胞分离及原代培养体系。OBJECTIVE： To provide a simple method for the isolation,purification and identification of human ovari- an cancer microvascular endothelial cells. METHODS： The specimens were collected from 21 cases of epithelial ovarian cancer in Shandong Cancer Hospital form 03-01-2012 to 10-31-2012. Collagenase digestion in combination with immu- nomagnetic sand method was used to separate, extract and purify human ovarian cancer microvascular endothelial cells. The obtained endothelial cells were identified by morphology observation,immunofluorescence and the formation of tubule- like structure. RESULTS： The purity in all obtained microvascular endothelial cells was higher than 95% and all the cells expressed endothelial cell specific, marker vWF, presented typical paving-stone sample characteristics and formated tubule- like structure in vitro. Endothelial cells attached in 24 hours in general and had completely integrated in 7-11 days and presented the contact inhibition phenomenon. Growth curve of cells showed ＂S＂ pattern using MTT method. CONCLU- SION： A reliable and relatively simple method is established for obtaining and culturing high purity,good vitality micr.o- vascular endothelial cell using immunomagnetic sand method.

关 键 词：卵巢肿瘤 上皮 内皮细胞 血管 荧光免疫测定 流式细胞术 原代培养 

分 类 号：R737.31[医药卫生—肿瘤]