人子宫内膜癌血管内皮细胞的分离鉴定及生物学特性检测  

Isolation, purification and characterization of endothelial cells from endometrial cancer and normal endometrial tissue

作　　者：杜雪莲[1] 盛修贵[1] 颜春晓[1] 刘婷[1] 路春华[1] 王聪[1] 于浩[1] 李庆水[1] 

机构地区：[1]山东省肿瘤医院妇瘤科,济南250117

出　　处：《中华肿瘤杂志》2012年第6期409-413,共5页Chinese Journal of Oncology

基　　金：基金项目：国家自然科学基金（30901713）;山东省科学技术攻关项目（2009GG10002010）;山东省自然科学基金（BS2010YY065）

摘　　要：目的建立人子宫内膜癌血管内皮细胞分离纯化及体外培养方法，观察所分离的人子宫内膜癌血管内皮细胞的生物学特性。方法采用优化的CD31单克隆抗体交联免疫磁珠法，对子宫内膜癌组织和正常子宫内膜组织标本进行血管内皮细胞分选、鉴定、纯度检测及体外培养。应用四甲基偶氮唑蓝（MTY）法、划痕实验、Transwell侵袭小室模型、Matrigel胶体外管腔形成实验，比较人子宫内膜癌血管内皮细胞和正常子宫内膜血管内皮细胞的增殖、迁移、侵袭和管腔形成能力。结果分选获得的两种血管内皮细胞纯度均〉95％，体外培养呈现典型的内皮细胞形态特征，表面均表达特异性标志物CD31和vW因子。MTT法检测结果显示，人子宫内膜癌血管内皮细胞和正常子宫内膜血管内皮细胞的增殖水平差异无统计学意义（P=0．173）。人子宫内膜癌血管内皮细胞和正常子宫内膜血管内皮细胞的划痕修复面积百分比分别为（37．54±5．63）％和（16．37±2．88）％，穿膜细胞数分别为（189±31）个／高倍视野和（131±22）个／高倍视野，形成的血管管腔样结构分别为（62±16）个和（35±11）个。与正常子宫内膜血管内皮细胞比较，人子宫内膜癌血管内皮细胞的划痕修复面积明显增加（P=0．006），穿膜细胞数显著增多（P=0．033），形成的血管管腔样结构明显增加（P=0．029）。结论采用优化的免疫磁珠分选法可高纯度地分离纯化出子宫内膜癌和正常子宫内膜组织中的血管内皮细胞。与正常子宫内膜血管内皮细胞比较，人子宫内膜癌血管内皮细胞的迁移、侵袭和血管生成能力明显增强。Objective To immunopurify human endometrial endothelial cells （HEEC） from fresh surgical specimens of endometrial cancers and normal endometrial tissues, and investigate their biological characteristics. Methods Endothelial ceils of endometrial cancers and normal endometrial tissues were isolated using anti-CD31 conjugated magnetic microbeads. The isolated endothelial cells were cultured in vitro and their origins were identified. Their angiogenic characteristics were observed by MTI＇, wound healing, Transwell cell invasion and tube formation assays. Results Flow cytometry revealed that the immunopurification technique yielded endothelial cell purity of 〉 95% in all samples. All purified HEEC were characterized as endothelial ceils on the basis of expression of the classical endothelial markers vWF and CD31 as shown by immunofluorescence examination. Although the tumor-associated HEEC didn＇t show more rapid proliferation than normal HEEC, they exhibited enhanced migration ability （P = 0.006）, potent invasiveness （P = 0. 033 ）, and elevated tube formation in vitro （ P = 0. 029 ）. Conclusions Human endometrial endothelial cells can be efficiently isolated from endometrial cancer and normal endometrial tissues by immunomagnetic methods. Tumor-associated HEEC exhibit enhanced migratory ability, potent invasiveness, and elevated tube formation in vitro.

关 键 词：子宫内膜肿瘤 内皮细胞 免疫磁珠 新生血管化 病理性 

分 类 号：R737.33[医药卫生—肿瘤]