黏液蛋白Mucins在胎盤發育過程的表現及其與子癇前症的關聯之研究

Abstract

胎盤是胎兒賴以生存的生命線。而胎盤內的子宮動脈的末端血管「螺旋動脈」在胎盤發育過程中，能否充分完成動脈的再鑄型(remodeling)，攸關胎盤功能的優劣及胎兒的發育是否正常。而螺旋動脈的再鑄型與絨毛外細胞型滋養層母細胞取代螺旋動脈的血管內皮細胞的程序有關，所以滋養層母細胞在子宮內膜(蛻膜)區是否可以正常地移行(migration)、侵襲(invasion)，滋養層母細胞最後能否靠近及進行螺旋動脈血管內侵襲是十分重要的。 由於黏液蛋白的性狀改變，被認為與細胞的生長、複製、分化、轉型(transformation)乃至於細胞的移動、侵襲有關。而一些胞膜型黏液蛋白在胎盤的表現特別多，所以我們假設黏液蛋白與滋養層母細胞的蛻膜內侵襲作用有關。 我們觀察主要的黏液蛋白(MUC1及MUC15等)在整個懷孕過程中不同時期的基因和蛋白質表現，組織中的分布及它們的可能角色。同時我們會以細胞生物學的研究方法，了解黏液蛋白對於細胞型滋養層母細胞的侵襲作用的影響。我們會用絨毛膜癌的細胞株(主要細胞成分就是滋養層母細胞)，給予transfection之後，在黏液蛋白overexpression的情況下，以matrigel invasion assay來觀察滋養層母細胞的侵襲作用會如何改變，之後再以gelatin zymography或其他研究來尋找可能的機轉。 結果顯示MUC15 在胎盤中的表現最高。MUC1及MUC15的mRNA和protein的表現會隨著週數增加而遞增。(P < 0.05). 組織免疫染色顯示MUC1及MUC15 protein在cytotrophoblasts和syncytiotrophoblasts都有表現，而在apical membrane of syncytiotrophoblasts又特別明顯. 此外，MUC1及MUC15 在蛻膜的腺體上皮細胞也都有表現。而MUC15的Overexpression 明顯降低 JAR and/or JEG-3 cells 在matrigel的invasion，此現象與tissue inhibitors of metalloproteinases (TIMP)-1 及 TIMP-2的mRNA expression增加有密切關係。如果以small interfering RNA來knockdown MUC15會逆轉上述現象 (P < 0.05)。有趣的是，我們在蛻膜區發現MUC1-positive及MUC1-negative兩種族群的extravillous trophoblasts, 而且MUC1-positive extravillous trophoblasts會隨週數增加而增加。另外，MUC1 overexpression明顯抑制JAR cells在matrigel的 invasion (P< 0.01)，而此現象與 MMP9 activity下降有關。在重度的子癇前症的胎盤，MUC1的表現也明顯增加。 整體上看來，在human placentas的MUC1及MUC15的differential expression應該與regulation of trophoblast invasion息息相關，所以也可能與preeclampsia的發生產生關聯。有關臨床的影響及將來針對病生理學的研究的建議也會陸續討論。

Trophoblast invasion is crucial for the development of normal placentas. One of the primary placental defect in preeclampsia and partly intrauterine fetal growth restriction is shallow invasion of the extravillous trophoblast into the decidua, which leads to incomplete spiral artery remodeling and inadequate uteroplacental perfusion. Mucins are highly glycosylated proteins expressed by epithelial cells of various organs. The membrane-bound mucins, which possess a single transmembrane domain and a highly conserved cytoplasmic tail, include MUC1, MUC3A, MUC4, MUC12, MUC13, MUC15, MUC16, MUC17, and MUC20. Among the known mucins, MUC1 is best studied and plays crucial roles in regulating many cellular properties, including cell proliferation, apoptosis, adhesion, and invasion. Although MUC1 has been found to be expressed by trophoblasts of various species, its expression in the human placenta throughout pregnancy and its potential role in trophoblast invasion remain unclear. MUC15 is a membrane-bound mucin and the mRNA of MUC15 has been detected by RT–PCR in various human tissues. So far, physiological functions of MUC15 have never been investigated. In the present study, we therefore investigated MUC1 and MUC15 expression in the human placenta and the effect on the invasion of trophoblast cells. MUC15 mRNA in human tissues was analyzed by Northern blot. MUC1 and MUC15 mRNA and protein in human placenta were detected by real-time RT-PCR and Western blot, respectively. The distribution of MUC1 and MUC15 was revealed by immunohistochemistry. The effects of MUC1 and MUC15 on trophoblast invasion in vitro were analyzed by matrigel invasion assay in human choriocarcinoma JAR and/or JEG-3 cells. The results showed that MUC15 was expressed most highly in human placenta. Both MUC1 and MUC15 mRNA and protein increased with gestational age (P < 0.05, first versus third trimester). Immunohistochemistry showed that MUC1 and MUC15 protein was expressed by both cytotrophoblasts and syncytiotrophoblasts, especially at the apical membrane of syncytiotrophoblasts. In addition, MUC1 and MUC15 were found to be present in the glandular epithelium of the decidua. Overexpression of MUC15 substantially decreased matrigel invasion of JAR and/or JEG-3 cells, which was closely associated with an increase in mRNA expression of tissue inhibitors of metalloproteinases (TIMP)-1 and TIMP-2. Knockdown of MUC15 with small interfering RNA significantly reversed these effects (P < 0.05). Interestingly, we found two populations of extravillous trophoblasts, MUC1-positive and MUC1-negative cells, in decidua. The numbers of MUC1-positive extravillous trophoblasts were increased during placental development. Furthermore, MUC1 overexpression significantly (P< 0.01) suppressed matrigel invasion of trophoblast-like JAR cells which was associated with a decrease in MMP9 activity assessed by gelatin zymography. Besides, MUC1 expression was significantly higher in the placenta of severe preeclampsia if compared with normal ones. It seems that differential expression of MUC1 and MUC15 in human placentas may play a critical role in the regulation of trophoblast invasion. They may play some role in the disease process of preeclampsia. The clinical implications and further suggestions for pathophysiology studies are discussed here.