Investigation of controlled human extravillous trophoblast invasion

人类滋养细胞分化为两种主要的细胞系：绒毛细胞和外绒毛细胞。绒毛滋养细胞覆盖在胎盘的绒毛膜绒毛上，其功能是从母体向胎儿输送氧气和营养物质。相反，胞外滋养细胞（EVT）侵入母体子宫，也向螺旋动脉迁移，将螺旋动脉转化为低阻力的大血管。EVT侵袭的许多方面与肿瘤细胞侵袭时发生的事件相似。然而，与肿瘤侵袭不同，EVT侵袭受到精确调控，空间上局限于子宫，时间上局限于妊娠早期。EVT侵袭的机制尚不清楚。因此，在本项目中，我们首先通过几种方法确定了在EVT中特异性表达的分子。此后，我们研究了每个分子在人类滋养细胞中的作用，特别是在它们的控制侵袭性中。我们培养小鼠抗人term chorion laeve（其含有丰富的EVT）的单抗，以鉴定EVT中表达的新分子。获得了几个与EVT反应的单抗克隆，并对其抗原进行了氨基酸测序、cDNA测序或免疫筛选。这些分析表明，整合素α 5、细胞角蛋白18和黑色素瘤细胞粘附分子（MCAM）在EVT中表达。此外，mAb 5-23还能识别200 kD的未知细胞表面蛋白。除了单抗分析外，我们还进行了cDNA微阵列分析，以鉴定妊娠晚期无创绒毛膜EVT与妊娠早期胎盘有创EVT中表达差异的基因。鉴定出几个功能未知的表达序列标签（EST）。其中，EST9主要在含有侵袭性EVT的妊娠早期胎盘中表达。用5'RACE测定cDNA的全长，得到的EST9 mRNA长度为6kb，编码了一个推测的40kD蛋白转录因子。EST9在人滋养细胞中的功能目前正在研究中。少

Human trophoblasts differentiate into two main cell lineages : villous and extravillous cells. Villous trophoblasts cover the chorionic villi of the placenta, their function being to transport oxygen and nutrients from the mother to the fetus. In contrast, extravillous trophoblasts (EVT) invade maternal uterus and also migrate up the spiral arteries, transforming them into large vessels of low resistance. Many aspects of EVT invasion are similar to events that occur during tumor cell invasion. However, unlike tumor invasion, EVT invasion is precisely regulated, confined spatially to the uterus and temporally to early pregnancy. The mechanism of controlled EVT invasion is not clarified well.Therefore in this project, we first identified molecules which specifically express in EVT by several methods. Thereafter, we examined the role of each molecule in human trophoblasts, especially in their controlled invasiveness.We raised mouse mAb against human term chorion laeve (its contains abunda … More nt EVT) to identify novel molecules which are expressed in EVT.Several mAb clones which react with EVT were obtained, and their antigens were analyzed by amino acid sequencing, cDNA panning, or by immunoscreening method. These analyses revealed that integrin alpha 5, cytokeratin 18, and melanoma cell adhesion molecule (MCAM) are expressed in EVT.In addition, mAb 5-23 recognized the unknown cell surface protein of 200 kD.Besides mAb analyses, we carried out cDNA microarray analysis to identify genes which is differently expressed in non-invasive EVT of 3rd trimester chorion laeve compared with still invasive EVT of 1st trimester placenta. Several expressed sequence tag (EST) of unknown function were identified. Among these EST, EST9 was predominantly expressed in 1st trimester placenta which contains invasive EVT.Full length cDNA was determined by 5'RACE, and resultant EST9 mRNA of 6kb encoded a putative transcription factor of 40kD protein. Functions of EST9 in human trophoblast are now under investigation. Less