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Physiological roles of placental leucine aminopeptidase/oxytocinase and its clinical application using molecular biological technique.

胎盘亮氨酸氨肽酶/催产素酶的生理作用及其利用分子生物学技术的临床应用。

基本信息

批准号：
12470341
负责人：
MIZUTANI Shigehiko
金额：
$ 5.5万
依托单位：
Nagoya University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2002
项目状态：
已结题

项目摘要

Placental leucine aminopeptidase (P-LAP), which is identical to cystine aminopeptidase, is the predominant and functional oxytocinase in the placenta and maternal serum. The aim of this study is to evaluate the clinical significance and application of P-LAP using molecular biological technique. We have succeeded in establishing the CHO cells producing recombinant soluble form of P-LAP. Recombinant P-LAP hydrolyses not only oxytocin and vasopressin, but also dynorphin A and enkephalin in the brain. Although further in vivo studies are required, recombinant P-LAP could be used to inhibit the labor. P-LAP exists in both membrane-bound and soluble forms, which means that the soluble form is released by a specific proteolytic cleavage. P-LAP secretase has a metalloprotease activity and recognizes the amino acid sequence of cleavage site to attack. We have isolated genomic clones containing the 5'-upstream region of the P-LAP gene. We demonstrated that transcription factors activating protein 2 (AP-2) and Ikaros cooperatively enhance P-LAP transcription in trophoblastic cells. Interleukin-lbeta (IL-1β) increased P-LAP activity and proteins. Semi-quantitative RT-PCR and Southern blotting showed that IL-1β also increased P-LAP mRNA, which was abrogated by prior exposure to cycloheximide. Luciferase assays did not reveal any regulatory regions that could explain IL-1β-induced P-LAP mRNA accumulation. These findings indicated that prolonged exposure to IL-1β induces P-LAP in the placenta, possibly via other de novo protein synthesis. Transmission immunoelectron microscopy revealed that P-LAP was expressed on the surface of apical microvilli of syncytiotrophoblast cells. During this study, we could obtain both P-LAP-deficient mice and P-LAP overexpressing rats. Now the studies employing these animals to evaluate the changes of the spontaneous and oxytocin-induced labor are on the way. The results would elucidate the roles of oxytocinase during pregnancy.

胎盘亮氨酸氨肽酶（P-AMP）与胱氨酸氨肽酶相同，是胎盘和母体血清中主要的功能性催产素酶。本研究的目的是应用分子生物学技术评价P-BMPs的临床意义和应用价值。我们已成功地建立了生产重组可溶性形式P-glutamate的CHO细胞。重组P-FSH不仅水解催产素和加压素，还水解脑中的强啡肽A和脑啡肽。尽管需要进一步的体内研究，但重组P-glycoprotein可用于抑制分娩。β-淀粉酶以膜结合和可溶形式存在，这意味着可溶形式通过特异性蛋白水解裂解释放。β-淀粉样蛋白分泌酶具有金属蛋白酶活性，并识别切割位点的氨基酸序列进行攻击。我们已经分离出含有P-gp基因5 ′上游区的基因组克隆。我们证明，转录因子激活蛋白2（AP-2）和Ikaros协同增强滋养层细胞中P-gp的转录。白细胞介素1 β（IL-1β）增加P-gp活性和蛋白质。半定量RT-PCR和Southern杂交结果显示，IL-1β还可增加P-gp mRNA的表达，而放线菌酮可抑制P-gp的表达。荧光素酶测定没有发现任何可以解释IL-1β诱导的P-LAP mRNA积累的调节区域。这些发现表明，长期暴露于IL-1β诱导胎盘中的P-CRP，可能通过其他从头蛋白质合成。透射免疫电镜显示，P-gp表达于合体滋养层细胞顶端微绒毛表面。在这项研究中，我们可以获得P-STAT缺陷小鼠和P-STAT过表达大鼠。目前，利用这些动物评价自然分娩和催产素诱导分娩的变化的研究正在进行中。这一结果将有助于阐明催产素酶在妊娠中的作用。