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首页> 外文期刊>Clinical and diagnostic laboratory immunology >Isolation and characterization of human placental trophoblast subpopulations from first-trimester chorionic villi.
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Isolation and characterization of human placental trophoblast subpopulations from first-trimester chorionic villi.

机译：早孕绒毛膜绒毛中人胎盘滋养层亚群的分离和鉴定。

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A method for the simultaneous preparation of highly enriched human placental trophoblast populations (villous and extravillous) from first-trimester placental villi (5 to 12 weeks) by using sequential trypsinization, percoll gradient centrifugation, and negative selection with anti-CD9 immunomagnetic separation is described. The purification method resulted in the isolation of four distinct trophoblast populations identified on the basis of morphology and phenotyping: (i) mononuclear villous cytotrophoblast cells which, through differentiation, become committed to syncytium formation; (ii) an extravillous trophoblast population which appeared as a "crazy pavement" and, with subsequent subculturing, differentiated morphologically to mononuclear cells; (iii) an extravillous trophoblast fraction which fused to form multinucleated trophoblast giant cells; and (iv) floating intermediate extravillous trophoblast cells which fused together to form cell clumps and which further differentiated to a mononuclear anchoring intermediate extravillous trophoblast. Short-term cultures of the freshly isolated cell fractions consisted of heterogeneous trophoblasts at different differentiation stages as determined by their varied biochemical and morphological properties. All the isolated trophoblast populations expressed the cytokeratin intermediate filament and the epithelium-specific cell-cell adhesion molecule E-cadherin. The isolated villous trophoblasts in culture expressed integrins alpha 6 and beta 4 and reduced levels of beta 1 subunits, whereas the proliferating extravillous trophoblast cultures expressed alpha 1, alpha 3, and alpha 5 and high levels of beta 1 integrin subunits, vitronectin receptor (alpha V beta 3/beta 5), and major histocompatibility complex class 1 molecules. Furthermore, the isolated trophoblast populations secreted metalloproteases (such as type IV collagenases [mainly 72- and 92-kDa enzymes, i.e., gelatinases A and B]) and urokinase plasminogen activator, as evaluated by substrate gel zymography. This method of isolation should facilitate in vitro studies of trophoblast proliferation, differentiation, invasion, virus interactions, cytokenesis, and immunology.
机译：描述了一种通过使用顺序胰蛋白酶消化，percoll梯度离心和使用抗CD9免疫磁分离进行阴性选择同时从妊娠前三个月的胎盘绒毛（5至12周）同时制备高度富集的人胎盘滋养细胞群体（绒毛和绒毛）的方法。纯化方法导致分离出根据形态学和表型鉴定出的四个不同的滋养层种群：（i）单核绒毛细胞滋养层细胞，通过分化而变成合胞体的形成; （ii）绒毛外滋养层种群，表现为“疯狂的铺路”，随后继代培养，形态上分化为单核细胞; （iii）绒毛外滋养层部分，融合形成多核滋养层巨细胞; （iv）漂浮的中间绒毛外滋养层细胞融合在一起形成细胞团块，并进一步分化为单核锚定的中间绒毛滋养层细胞。新鲜分离的细胞部分的短期培养物由处于不同分化阶段的异质滋养细胞组成，这由它们的不同生化和形态特性决定。所有分离的滋养细胞群体均表达细胞角蛋白中间丝和上皮特异性细胞粘附分子E-钙粘着蛋白。培养物中分离出的绒毛滋养层细胞表达整合素α6和β4，β1亚基水平降低，而增殖的绒毛外滋养层细胞表达α1，α3和α5以及高水平的β1整合素亚基，玻连蛋白受体（ V beta 3 / beta 5）和主要的组织相容性复合物1类分子。此外，通过底物凝胶酶谱评估，分离的滋养细胞群体分泌金属蛋白酶（例如IV型胶原酶[主要是72-kDa和92-kDa酶，即明胶酶A和B）和尿激酶纤溶酶原激活剂。这种分离方法应有助于对滋养层细胞增殖，分化，侵袭，病毒相互作用，细胞分裂和免疫学进行体外研究。

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