骨橋蛋白在小鼠胚胎之著床扮演之角色

在人工協助生殖技術的治療中，提升胚胎的著床(implantation)一直是難以突破的關鍵因素。由於參與著床的因子繁多，過去許多文獻指出，骨橋蛋白(osteopontin，OPN) 可增加細胞與細胞之間的結合能力，以及與細胞外基質(extracellular matrix)的訊息傳遞與細胞移行(cell migration)。研究陸續發現，人類子宮內膜上皮細胞於分泌期時，內膜腺體、免疫細胞及分泌物發現OPN的表達及分泌。在著床關鍵期(implantation window)，OPN的細胞膜接受器αvβ3 (integrin receptor)會大量表現在子宮內膜上皮，與子宮的胚胎接受度有高度相關性。因此，我們推測OPN可促使胚胎著床，本論文的主要目的是探討OPN如何調控胚胎著床於子宮內膜之分子機制。我們利用Matrigel invasion chamber系統體外培養小鼠胚胎，實驗結果顯示在囊胚期的胚胎培養時添加OPN培養後，相較於對照組其胚胎著床的機率有明顯提升1.87倍。而αvβ3抑制劑(anti-αvβ3 antibody)的添加，則降低OPN的促胚胎著床作用。我們利用寡核苷酸微陣列分析法(Oligonucleotide microarray)分析經由100 nM OPN添加培養的胚胎所影響的基因表現。經由結果分析後，有60個基因於OPN添加培養後使得表現量顯著性增加(Normalized Expression Ratio，NER＞5，p<0.05)以及51個表現量顯著性減少的基因(NER< 0.5，p<0.05)。在微陣列(microarray)分析中，表現量增加的基因中Regulators of G protein Signaling 2 (RGS2)有44.2倍上升。其他增加的基因中，如Wilms tumor homolog (Wt1) 有47.7倍上升; growth differentiation gene (GDF)基因有30.6 倍的增加；lactate dehydrogenase 2(Ldh2) 基因有26.9 倍的增加。特別RGS2基因，過去研究中有被提出在著床上扮演著重要角色。經由以real-time quantitative PCR 的確認下，證明了加入100nM OPN 添加於胚胎培養液中會促使得胚胎的上RGS2的mRNA 表現量較控制組增加77.7 ± 4.8 倍，而在anti-αvβ3 Ab 的添加作用下會抑制胚胎降低Rgs2的mRNA表現量，下降至控制組的7.6 ± 1.3 倍。再以Matrigel進行體外模擬著床分析，當胚胎給予Rgs2 inhibitor後再添加以100nM OPN處理，發現黏著於Matrigel 的比率會明顯下降( 40.82% vs. 12.5%)。由此，我們確認OPN經由Rgs2增加胚胎著床率。在以胚胎滋養層細胞(JAR)為實驗模式進行細胞移行(cell migration)實驗分析中，我們發現OPN可經由與αvβ3 integrin的結合活化RGS2訊息路徑，促進 JAR細胞移行。進一步分析細胞內ATP含量也增加1.63倍。經由細胞生物能量儀分析，發現OPN會增加JAR細胞耗氧率(oxygen consumption rate，OCR)及乳酸的產生率(extracelluar acidification rate, ECAR)。因此我們確認OPN可以增進細胞的能量代謝能力，促進細胞移行，進而增加胚胎的著床能力。

關鍵字

骨橋蛋白；胚胎；著床；骨橋蛋白細胞膜接受器；寡核苷酸微陣列分析法

並列摘要

The improvement of the implantation rate is an important issue in the assisted reproductive technology. The successful implantation is initiated via blastocyst-endometrium interaction and delicately regulated by multiple factors. Osteopontin (OPN) plays a role as a mediator of cell-cell, cell-extracellular matrix (ECM) communication and invasion. Studies show that OPN is a major constituent of the uterine-placental microenvironment with influence as a component required for adhesion and signal transduction. Several evidence show that OPN binds to αvβ3-integrins. In the implantation window, αvβ3 suddenly increased in endometrium that highly correlated with embryo implantation. The aim of this study is to discuss whether OPN can improve embryo implantation ability. By mating 8-wk-old wild-type female mice with 16-wk-old wild-type male mice, the 4 to 8 cell-embryos were collected and analyzed embryo developing competency. Using matrigel invasion chamber, the developing competency that embryos underwent hatching out and implantation were analyzed. When OPN supplemented in the culture medium, it enhanced the rate of attachment on the matrigel (the in vitro implantation rate) for 1.69-folde. Moreover, the implantation rate was inhibited by anti-αvβ3 antibody (Ab). To gain insights into the molecular mechanisms, we identified the candidate genes involved in this process including 60 significantly up-regulated genes (Normalized Expression Ratio, NER＞5, p<0.05) and 51 down-regulated genes (NER＜0.5, p<0.05) by oligonucleotide microarray. The up-regulated genes were Wt1 (47.7 folded increase), RGS2 (44.2), GDF9 (30.6), Btg4 (25),and Ldh (26.9). Content of the regulator of G-protein signaling protein 2 (RGS2) mRNA was found to be up-regulated to 44.2 fold, and we further more confirmed with real-time quantitative PCR analysis, RGS2 has 77.7 times fold-up compare with control. The RGS2 mRNA were reduced by 7.66-fold by anti-αvβ3 Ab when OPN was treated 100nM. The rate of hatching out and implantation were reduced by use of anti-αvβ3 Ab or RGS2 inhibitors. In addition, the human choriocarcinoma JAR cell line was used to perform in vitro cell migration. By scratch assay, OPN promoted-increased the JAR cell migration. The use of anti-αvβ3 Ab or RGS2 inhibitor with OPN co-treated attenuate the implantation and migration ability. Moreover, OPN enhanced ATP biosynthesis (1.63-folded increase). In the bioenergetic characterization, the increased oxygen consumption rate (OCR) and lactate production (extracelluar acidification rate, ECAR) were found in the OPN treatment. We concluded that the OPN can enhance cell bioenergetic metabolism and promote embryo implantation and developmental competency via interacting with αvβ3 integrin and activate RGS2 signaling pathway.

並列關鍵字

Osteopontin ； embryo ； implantation ； αvβ3 integrin receptor ； Ologonucleotide microarray

參考文獻

Anger, T., Grebe, N., Osinski, D., Stelzer, N., Carson, W., Daniel, W.G., Hoeher, M., and Garlichs, C.D. (2008). Role of endogenous RGS proteins on endothelial ERK 1/2 activation. Experimental and molecular pathology 85, 165-173.

Aplin, J.D. (2006). Embryo implantation: the molecular mechanism remains elusive. Reproductive biomedicine online 13, 833-839.

Armant, D.R., Wang, J., and Liu, Z. (2000). Intracellular signaling in the developing blastocyst as a consequence of the maternal-embryonic dialogue. Seminars in reproductive medicine 18, 273-287.

Campbell, S., Swann, H.R., Aplin, J.D., Seif, M.W., Kimber, S.J., and Elstein, M. (1995). CD44 is expressed throughout pre-implantation human embryo development. Hum Reprod 10, 425-430.

Casals, G., Ordi, J., Creus, M., Fabregues, F., Carmona, F., Casamitjana, R., and Balasch, J. (2010). Osteopontin and alphavbeta3 integrin as markers of endometrial receptivity: the effect of different hormone therapies. Reproductive biomedicine online 21, 349-359.

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骨橋蛋白在小鼠胚胎之著床扮演之角色

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