Sharma R K
Department of Anatomy and Cell Biology, University of Kansas Medical Center, Kansas City 66103, USA.
In Vivo. 1998 Mar-Apr;12(2):209-17.
The specialized interaction between embryonic and maternal tissue is unique to mammalian development. This interaction begins with the invasion of the uterus by the first differentiated embryonic cells, the trophoblasts, and culminates in formation of the placenta. Because of their highly specialized behavior invasive cells must attach to the extracellular matrix proteins, secrete proteinases, capable of degrading matrix, and migrate through the degraded matrix; invasion is partially dependent on the proteinase activity of the cells. The objective, therefore, was to study a vitro system to examine the mechanism(s) of trophoblast cell invasion and its relationship to proteinases. Since little is known about the actual mechanism(s) involved. The mouse trophoblast cell lines established from placentas of different gestational ages were chosen to study their invasive properties in vitro. To begin to understand the biochemical basis of this behavior, the chromogenic assay and the substrate gel technique was used to analyze the cell associated and secreted plasminogen activators. All lines secrete and synthesize both urokinase-type (uPA) and tissue-type (tPA) plasminogen activators. The most invasive line SM9-2, derived from mid-gestation (day 9) placenta showed the highest enzymatic activity in the conditioned medium (CM), whereas in cell extract (CE) SM-10 line derived from late gestation placenta had the highest PAs activity. Four forms of secreted PAs in CM were of 79, 72, 43 and 35 kDa molecular weights, whereas in CE only 79 kDa molecular weight form of PA was detected using substrate SDS-PAGE gels. Additional observations from cells cultured on Marrigel Invasion Chambers also showed secretion of PAs by noninvading and invading cells in a biphasic pattern suggest the involvement of these enzymes in the extracellular proteolysis. The expression of matrix metalloproteinase gelatinase B (MMP-9) and tissue inhibitor of metalloproteinase (TIMP-1) were examined by RT-PCR in all the lines, however MMP-9 and TIMP-1 signals were strongly expressed in SM9-2 and SM-10 lines respectively. CM and CE were characterized by gelatin zymography, and the proteinases secreted by these cells in CM were confirmed to be metalloproteinases with approximate molecular masses between 52 to 92 kDa. Proteinases secreted by noninvading and invading cells cultured on Matrigel Invasion Chambers were not identical suggesting that specialized, temporally regulated metallopro-teinases are involved in trophoblast invasion. Trophoblast cell invasion in Matrigel Invasion Chambers was significantly inhibited in all the lines by using 1, 10-phenanthroline, an inhibitor of metalloproteinases. The results indicated that mouse trophoblast cells have matrix--degrading capabilities through metalloproteinase activity. Similar metalloproteinase activity has been reported to be necessary for human trophoblast invasion, suggesting a similar mechanism of implantation. Trophoblast culture system described here should be useful in studying some of the early events in human placentation.
胚胎组织与母体组织之间的特殊相互作用是哺乳动物发育所特有的。这种相互作用始于最早分化的胚胎细胞——滋养层细胞对子宫的侵入，并最终形成胎盘。由于其高度特殊的行为，侵袭性细胞必须附着于细胞外基质蛋白，分泌能够降解基质的蛋白酶，并穿过降解后的基质进行迁移；侵袭部分依赖于细胞的蛋白酶活性。因此，目的是研究一种体外系统，以检查滋养层细胞侵袭的机制及其与蛋白酶的关系。由于对实际涉及的机制知之甚少，所以选择从不同妊娠年龄胎盘建立的小鼠滋养层细胞系来研究其体外侵袭特性。为了开始理解这种行为的生化基础，采用发色底物法和底物凝胶技术分析细胞相关和分泌的纤溶酶原激活剂。所有细胞系均分泌并合成尿激酶型（uPA）和组织型（tPA）纤溶酶原激活剂。侵袭性最强的细胞系SM9-2源自妊娠中期（第9天）胎盘，其条件培养基（CM）中的酶活性最高，而在细胞提取物（CE）中，源自妊娠晚期胎盘的SM-10细胞系的纤溶酶原激活剂活性最高。CM中分泌的四种形式的纤溶酶原激活剂分子量分别为79、72、43和35 kDa，而在CE中，使用底物SDS-PAGE凝胶仅检测到79 kDa分子量形式的纤溶酶原激活剂。在基质胶侵袭小室上培养细胞的其他观察结果也显示，非侵袭性和侵袭性细胞均以双相模式分泌纤溶酶原激活剂，这表明这些酶参与细胞外蛋白水解。通过RT-PCR检测了所有细胞系中基质金属蛋白酶明胶酶B（MMP-9）和金属蛋白酶组织抑制剂（TIMP-1）的表达，然而MMP-9和TIMP-1信号分别在SM9-2和SM-10细胞系中强烈表达。通过明胶酶谱法对CM和CE进行了表征，这些细胞在CM中分泌的蛋白酶被确认为分子量约在52至92 kDa之间的金属蛋白酶。在基质胶侵袭小室上培养的非侵袭性和侵袭性细胞分泌的蛋白酶并不相同，这表明特殊的、受时间调控的金属蛋白酶参与了滋养层细胞的侵袭。使用金属蛋白酶抑制剂1,10-菲咯啉可显著抑制所有细胞系在基质胶侵袭小室中的滋养层细胞侵袭。结果表明，小鼠滋养层细胞通过金属蛋白酶活性具有降解基质的能力。据报道，类似的金属蛋白酶活性对于人类滋养层细胞侵袭也是必需的，这表明存在类似的着床机制。这里描述的滋养层细胞培养系统对于研究人类胎盘形成的一些早期事件应该是有用的。
