人静脉畸形内皮细胞的分离培养和鉴定

Isolation, culture and identification of human venous malformation endothelial cells

目的 建立一个稳定的人血管畸形内皮细胞体外培养体系。方法 取人静脉畸形组织块接种于铺有明胶底层的培养瓶中进行原代及传代培养。通过早期去除组织块、机械刮除及酶消化法纯化内皮细胞。对培养的细胞进行形态学观察及免疫组化染色等一系列细胞定性研究。结果 获取的内皮细胞可连续传代 4～ 5代 ,成活 4 0～ 5 0d ;生长于玻璃、塑料上的细胞呈铺路石状 ;生长于明胶底层上的细胞可形成毛细血管样结构 ;电镜下可见细胞具有Weibel Palade小体特征性超微结构 ;CD3 4 及vWF免疫组化染色阳性 ,α SMA染色阴性。结论 应用此培养方法可获得纯化的血管畸形内皮细胞 ,且细胞可连续传代培养。培养的血管畸形内皮细胞可用于体外研究血管畸形的生物学行为。

Objective To establish a method of culturing endothelial cells (EC) from human vascular malformation. Methods Venous malformation specimens obtained from the patient undergoing oral surgery were plated into glass, plastic and gelatin coated dishes. Pure cultures of human vascular malformation endothelial cells (VMEC) were isolated by the ways of discarding the tissues at early stage of primary culture, scraping and trypsinizing. Morphological characteristics were studied under phase contrast microscope and electron microscope (EM), and determined by immunohistochemistry. Results The cells were pure and could be maintained in culture up to 4～5 passages, or 40～50 days. VMEC formed contact inhibited 'cobblestone' monolayer on glass and plastic , and capillary like'tubes' on gelatin. EM revealed that there were Weibel Palade bodies in the culture cells. The cells showed positive staining for CD 34 , vWF, and negative for α SMA. Conclusions The culture technique for growing VMEC has been established. And these cells can provide a useful tool for studying biological characteristics of human vascular malformation in vitro.