探討O-linked N-acetyl-glucosamine (O-GlcNAc)於子癲前症之胎盤的表現量分析

Abstract

(一) 背景及研究目的 子癲前症定義為妊娠20周後發生的母體高血壓合併尿蛋白或其他器官的功能異常。根據文獻，全球發生率約2-10%；在台灣婦女的族群，其發生率雖落在2-3%，但隨台灣孕產婦生產平均年齡上升，高血壓族群比例增加，子癲前症也有逐年上升的趨勢。尤其是早發性子癲前症（發生在妊娠34周前），不僅容易導致新生兒極度早產(extremely preterm birth)，且有較高比例發生母體併發症、胎兒生長遲滯 (fetal growth restriction)或胎死腹中(Intrauterine fetal death)。 子癲前症的病理機轉與異常的胎盤血管生成相關。臨床上可在超音波上觀察到阻力較大的子宮動脈血流，而組織切片上則可觀察到較細的螺旋血管（spiral arteries）以及較淺的滋養層侵襲（trophoblast invasion），進而影響胎盤供應及胎兒生長；而胎盤血管生成（placenta vasculogenesis）會受到氧化壓力（oxygen tension) 的影響。曾有動物研究顯示，胎盤對於缺氧（hypoxia）的反應與O-GlcNAcylation 程度有正相關。 O-linked N-Acetylglucosamine (O-GlcNAc) 接上細胞核、細胞質、或者粒線體上的某些特定蛋白質上的胺基酸serine (Ser) 以及 threonine (Thr) 的過程稱為O-GlcNAcylation，屬於一種轉譯後修飾（post-translational modification）。其過程受O-GlcNAc transferase (OGT)及O-GlcNAcase (OGA) 兩種酵素調控。在動物實驗顯示，在OGA-/-的實驗鼠胎盤組織，O-GlcNAcylation的程度增加，會下調(downregulate) OGT；而OGA 缺乏也會導致異常的胎盤生長。另有研究顯示O-GlcNAc過度表現(overexpression)與胎盤功能異常(placenta dysfunction)相關。然而，在子癲前症的人類胎盤研究上，目前的文獻仍有限。 此實驗的目的主要是分析O-GlcNAc在子癲前症及其周數對照的非子癲前症於人類胎盤表現上是否有差異、其在胎盤表現之組織學分佈；進一步分析於人類胎盤OGT 和 OGA兩種調控酵素蛋白於兩組間表現量的差異，探討其表現量差異的可能原因分析，以期對子癲前症的病理機轉有更進一步的了解。 (二) 研究設計 試驗的執行期間為 2019年1月24日- 2020年12月31日，取得病患同意書後，蒐集母體產檢過程之臨床資料如血壓、尿蛋白、生產週數、母體合併症、以及母胎預後。於分娩後(自然產或剖腹產)，進行胎盤之採樣，利用免疫組織化學染色(immunohistochemistry)、西方墨點法(western blot)，分析O-GlcNAc, OGT, OGA 於子癲前症以及非子癲前症產婦於胎盤之表現量及組織學上之分佈；免疫組織化學染色的結果使用H-score量化。使用統計方法為 independent sample t test及Mann-Whitney U test，p<0.05視為統計上有顯著意義。 (三) 研究結果 臨床結果發現，早發性子癲前症與對照組平均生產周數無顯著差異，但胎兒平均出生體重在早發性子癲前症較輕，顯示於早發性子癲前症有較高比例之胎兒生長遲滯。在免疫染色切片發現：O-GlcNAc在早發性子癲前症之胎盤（28-33+6周, n=10）對照周數相當之非子癲前症胎盤(n=5)在蛻膜層(decidual layer)及絨毛膜層 (chorionic villi)其染色顯著地較強( p=0.014*)，其中以在蛻膜細胞(decidual cell)其色差異最為顯著 (IHC 2+-3+ vs. IHC 0-1+)；而在晚發性子癲前症之胎盤（34-36+6周, n=2）對照周數相當之非子癲前症胎盤(n=2)，O-GlcNAc於兩組的染色差異則較不明顯 (IHC皆為 1+-2+)。就OGT 及OGA兩個調控的蛋白染色發現：其結果與O-GlcNAc相似，在早發性子癲前症之胎盤OGT及OGA的染色在蛻膜層(decidual layer)以及絨毛膜層 (chorionic villi)其染色皆明顯地較強(OGT: p=0.001**; OGA: p=0.001**)，尤其在蛻膜細胞(decidual cell)其差異最為顯著 (IHC 2+-3+ vs. IHC 0-1+)；而在晚發性子癲前症之胎盤（35-36周）OGT及OGA在實驗組及對照組的表現皆偏淡（IHC 1+），兩組無明顯差異。總結，O-GlcNAc 以及其兩個調控蛋白OGT和OGA在早發發性子癲前症之胎盤表現量顯著增加。

Background Preeclampsia is a maternal syndrome of hypertension with proteinuria or organ dysfunction after 20 weeks of gestation, which characterized by the insufficient trophoblast invasion and inadequate uteroplacental blood flow. The ischemia leads to placental tissue hypoxia, which exacerbates the poorly developed fetoplacental vasculature, resulting in the compromised oxygen and nutrient delivery to the fetus. The O-linked N-Acetylglucosamine (O-GlcNAc) attached to serine (Ser) or threonine (Thr) residues of nucleocytoplasmic and mitochondrial proteins is a post-translational modification. The O-GlcNAcylation process is reversible and dynamically controlled by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). It has been reported that increased O-GlcNAcylation may give rise to vascular dysfunction in hypoxia condition in animal models, suggesting the association between O-GlcNAc cycling and hypoxia-related diseases. Preeclampsia is featured with poor placental vascularity, abnormal trophoblast invasion, and placental hypoxia, and the pathogenesis could be altered by O-GlcNAcylation. Therefore, the aim of this study is to compare expression and distribution of O-GlcNAc and O-GlcNAc cycling enzymes in the human placenta of preeclampsia with those of non-preeclampsia. Methods We recruited gestational age paired pregnant women with preeclampsia and non-preeclampsia, who were singleton pregnancy. At delivery, placenta tissue was collected and subjected to immunohistochemistry (IHC) and western blot analysis, for the expression of O-GlcNAc and O-GlcNAc cyclic enzymes, including OGT and OGA. Results Between the gestational age paired groups, fetal growth restriction (FGR) was significantly observed in the early-onset preeclampsia group (28-33 weeks), compared with non-preeclampsia group. Meanwhile, IHC stain revealed significantly higher expression levels of O-GlcNAc, OGT, and OGA in the placenta of early-onset preeclampsia in both decidua and chorionic villi compared with that in the non-preeclampsia group (p-value: O-GlcNAc 0.012**, OGT: 0.004**, OGA: 0.004**). The higher expression was especially prominent in decidual cells. However, IHC showed that the difference in the O-GlcNAc expression between late-onset preeclampsia (35-36 weeks) and gestational age paired non-preeclampsia group was not significant in both decidual and chorionic villi layers. Both OGT and OGA showed weak expression in the placenta of late-onset preeclampsia and non-preeclampsia group. Conclusion The O-GlcNAc and O-GlcNAc cycling enzymes were increased in the placenta of women with early-onset preeclampsia.