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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.

胎盘亮氨酸氨基肽酶(P-LAP)是胎盘和母体血清中最主要和功能最强的催产素酶，与胱氨酸氨基肽酶相同。本研究的目的是应用分子生物学技术评价P-LAP的临床意义和应用。我们成功地建立了生产重组可溶性P-LAP的CHO细胞。重组P-LAP不仅能降解催产素和加压素，还能降解大脑中的强啡肽A和脑啡肽。虽然还需要进一步的体内研究，但重组P-LAP可以用于抑制分娩。P-LAP以膜结合和可溶性两种形式存在，这意味着可溶性形式是通过特定的蛋白水解性切割释放的。P-LAP分泌酶具有金属蛋白酶活性，识别切割位点的氨基酸序列进行攻击。我们已经分离到了含有P-LAP基因5‘-上游区域的基因组克隆。我们证明了转录因子激活蛋白2(AP-2)和Ikaros协同增强滋养层细胞中P-LAP的转录。白介素1β(IL-1β)可增加P-LAP活性和蛋白表达。半定量逆转录聚合酶链式反应和Southern blotting结果显示，IL-1β还能增加P-LAP基因的表达，而这一作用可被放线菌素预先作用于放线菌酮所抑制。荧光素酶检测没有发现任何可以解释IL-1β诱导的P-LAP基因积聚的调控区域。这些发现表明，长期暴露于IL-1β可诱导胎盘中的P-LAP，可能是通过其他从头合成蛋白质来实现的。透射免疫电子显微镜显示P-LAP表达于合体滋养层细胞顶端微绒毛表面。在本研究中，我们既可以获得P-LAP缺陷小鼠，也可以获得P-LAP过表达大鼠。现在，利用这些动物来评估自然分娩和催产素引产的变化的研究正在进行中。这一结果将阐明催产素酶在妊娠过程中的作用。