Iruela-Arispe M L, Rodriguez-Manzaneque J C, Abu-Jawdeh G
Department of Pathology, Harvard Medical School, Boston, MA, USA.
Microcirculation. 1999 Jun;6(2):127-40.
To develop a reliable method for the isolation and longterm culture of microvessel endothelial cells from human endometrium and to evaluate their response to angiogenic growth factors and steroid hormones in comparison to endothelial cells derived from other organs.
Endometrial tissue from hysterectomy specimens were digested sequentially with collagenase and trypsin, cultured for 24 h, then selected by adhesion to anti-CD-34 coated magnetic beads. Alternatively, anti-CD-34-coated beads could also be substituted by Ulex europaeus agglutinin-1, anti-PECAM, or anti-E-selectin-coated beads. Characterization of the isolated cultures included expression of endothelial cell markers, regulation of E-selectin in response to TNF-alpha, proliferative response to angiogenic growth factors, and expression of progesterone and estrogen receptors. We also analyzed the relative binding affinity of VEGF on endometrial endothelial cells in comparison to other endothelial cell types.
Selection on anti-CD-34-coated beads eliminated contaminating cells and resulted in a homogeneous population of human endometrial endothelial cells (HEEC), as assessed by expression of PECAM, von Willebrand's factor, and uptake of acetylated-LDL. HEEC also upregulated E-selectin in response to TNF-alpha in a manner similar to that seen for other endothelial cell types. Expression of progesterone and estrogen receptor was revealed by immunocytochemistry and RT-PCR consistently until passage 5. Endometrial endothelial cells were more responsive to growth stimulation by VEGF than were dermal endothelial cells isolated under similar conditions. Further characterization indicated that VEGF bound more avidly to HEEC than to other endothelial cell types.
Human endometrial endothelial cells were isolated to homogeneity by a two-part protocol and successfully passaged under culture conditions similar to those used for other endothelial cell types. The HEEC were very responsive to VEGF growth-stimulation likely due to elevated affinity, or increased levels of, KDR and FLT-1 on the cell surface. These results indicate that HEEC are capable of maintaining a mature phenotype in culture and might provide a model for understanding the response of these cells to the recurrent cycles of proliferation imposed on the endometrium during menstruation.
建立一种可靠的从人子宫内膜中分离和长期培养微血管内皮细胞的方法，并与源自其他器官的内皮细胞相比，评估其对血管生成生长因子和甾体激素的反应。
子宫切除标本的子宫内膜组织先用胶原酶和胰蛋白酶依次消化，培养24小时，然后通过与抗CD-34包被的磁珠黏附进行筛选。或者，抗CD-34包被的磁珠也可用荆豆凝集素-1、抗PECAM或抗E-选择素包被的磁珠替代。对分离培养物的特性鉴定包括内皮细胞标志物的表达、对肿瘤坏死因子-α（TNF-α）反应时E-选择素的调节、对血管生成生长因子的增殖反应以及孕酮和雌激素受体的表达。我们还分析了与其他内皮细胞类型相比，血管内皮生长因子（VEGF）对子宫内膜内皮细胞的相对结合亲和力。
通过抗CD-34包被的磁珠筛选消除了污染细胞，并产生了均一的人子宫内膜内皮细胞（HEEC）群体，这通过PECAM、血管性血友病因子的表达以及乙酰化低密度脂蛋白（LDL）的摄取来评估。HEEC对TNF-α的反应中上调E-选择素的方式与其他内皮细胞类型相似。免疫细胞化学和逆转录-聚合酶链反应（RT-PCR）一致显示，直到第5代，孕酮和雌激素受体的表达一直存在。与在相似条件下分离的真皮内皮细胞相比，子宫内膜内皮细胞对VEGF的生长刺激反应更敏感。进一步的特性鉴定表明，VEGF与HEEC的结合比与其他内皮细胞类型更紧密。
通过两步方案将人子宫内膜内皮细胞分离至均一状态，并在与用于其他内皮细胞类型相似的培养条件下成功传代。HEEC对VEGF生长刺激反应非常敏感，这可能是由于细胞表面KDR和FLT-1的亲和力升高或水平增加。这些结果表明，HEEC能够在培养中维持成熟表型，并可能为理解这些细胞对月经期间子宫内膜反复增殖周期的反应提供一个模型。
