鄰苯二甲酸酯對人類卵巢顆粒細胞及子宮內膜間質性幹細胞之影響

鄰苯二甲酸酯類(phthalates)，俗稱為塑化劑，可視為一種環境毒素，例如鄰苯二甲酸甲苯基丁酯(butyl benzyl phthalate，縮寫：BBP)是聚氯乙烯製程中相當常見的添加物，很容易殘留在日常使用的塑膠用品上。愈來愈多的證據顯示塑化劑可以干擾正常內分泌及代謝功能、影響組織發育，並且影響生育功能，但塑化劑如何影響生育功能的機轉還不是很清楚。人類生育的啟源來自於具有功能的卵子和精子受精後，形成健康的胚胎，在適當的著床時間，在功能正常的子宮內膜著床。卵子的品質可能受到濾泡內顆粒細胞所影響，而著床成敗的關鍵則可能在子宮內膜成長及分化的品質。因此我們從塑化劑對卵巢顆粒細胞，及子宮內膜間質性幹細胞可能造成影響及其機轉進行探討，以期從分子及基因的層次來探討塑化劑對生育婦女的危害。首先，我們以不孕症患者接受試管嬰兒療程時，取卵過程中所取得之卵巢顆粒細胞，以及永生之顆粒細胞株HO23進行BBP對aryl hydrocarbon receptor (AhR)所造成之影響。1μM濃度之BBP會明顯增加AhR、aryl hydrocarbon receptor nuclear translocator (ARNT)及cytochrome-P450(CYP)1B1在 HO23細胞株的mRNA及蛋白質的表現，若以3’4’-dimethoxyflavone (3’4’-DMF)或AhR siRNA來處理細胞會明顯減少AhR和CYP1B1的表現，但不會影響到CYP1A1，所以CYP1A1的表現應該不受AhR所調控。BBP造成AhR融合蛋白會累積在細胞核邊緣，經由免疫沈澱法分析可以發現AhR和ARNT的異源二聚體普遍存在細胞核中。染色質免疫沉澱測定顯示出BBP作用在CYP1B1，而不是CYP1A1。經BBP處理之後的HO23，細胞壞死的比例明顯增加，但3’4’-DMF、AhR siRNA或CYP1B1 siRNA的抑制作用可以阻斷這個現象。這些研究結果顯示出BBP誘導之HO23細胞壞死是依靠AhR及CYP1B1來完成。子宮內膜間質性幹細胞(EN-MSCs)的分化能力會因為BBP的作用而降低，使用cDNA microarrays來分析BBP在調節EN-MSCs的基因表現，並用IPA軟體來解讀受影響的基因及生物功能。從骨骼肌肉異常、細胞形態及組織發展等三個不同族群的影響基因，可以歸納出PITX2 (Paired-like homeodomain transcription factor 2)基因是最重要的交集。BBP會減少PITX2的轉錄，並提高microRNA miR137的表現，因此預測miR-137可以從上游降調PITX2的表現。實驗結果顯示BBP經由影響miR-137作用在PITX2 mRNA 3’端的未轉譯區域來影響PITX2的表現，PITX2的降調也會在EN-MSCs減少MyoD的轉譯量。這些實驗結果顯示出BBP會經由上調miR-137的表現量來減少EN-MSCs的肌細胞分化能力，讓我們瞭解到子宮內膜的分化可能受到塑化劑的影響，因此可能會影響子宮內膜對胚胎的著床能力，暗示環境荷爾蒙對生育能力具有之潛在危機。

關鍵字

子宫內膜間質性幹細胞；小分子核糖核酸-137 ；芳香烴受體；顆粒細胞；塑化劑；鄰苯二甲酸甲苯基丁酯；類成對同源框轉錄因子2

並列摘要

Phthalate, an environmental toxin, has been considered as an endocrine-disrupting chemical. Growing evidence has demonstrated links between endocrine-disrupting chemicals, tissue development, and reproductive physiology, but the mechanisms of reproductive disturbance regulated by these environmental factors are unclear. As we known, a successful pregnancy is basic on healthy eggs fertilized by healthy sperms to form functional embryos, then implanted into mature endometrium at the time of implantation window. Granulosa cells in follicles and around human eggs maybe play an important role in oocyte maturation. The endometrial quality regulated by estrogen and progesterone imply another important factor in implantation. According to these reasons, we investigated the signaling pathway of the arylhydrocarbon receptor (AhR) on HO23 cells (immortalized human granulosa cells (hGC)) mediated by benzyl–butyl-phthalate (BBP). Then, we investigated the effects BBP on human endometrial mesenchymal stem/stromal cells (EN-MSCs) differentiation and identified a novel signaling pathway. BBP (1 ?嵱) significantly increased the mRNA and protein levels of AhR, aryl hydrocarbon receptor nuclear translocator (ARNT) and cytochrome-P450 (CYP)1B1 in HO23 cells. Treatment with 3’,4’-dimethoxyflavone (3’,4’-DMF) or AhR siRNA significantly reduced AhR and CYP1B1, but CYP1A1 was not affected by 3’,4’-DMF or AhR siRNA, suggesting that increases in CYP1A1 may not regulated by AhR. BBP induced the AhR fusion protein to localize and accumulate around the nucleus, and AhR heterodimerization with ARNT was observed in the nucleus by immunoprecipitation. Chromatin immunoprecipitation and reporter assays revealed the effect of BBP on CYP1B1, but not CYP1A1. Necrosis was significantly increased in HO23 cells after BBP treatment, and 3’,4’-DMF, AhR siRNA or CYP1B1 siRNA knockdown blocked this phenomenon. These data suggest that BBP-induced HO23 cell necrosis is AhR and CYP1B1 dependent. Differentiation of EN-MSCs decreased after administration of BBP. We analyzed BBP regulation of gene expression in EN-MSCs using cDNA microarrays and Ingenuity Pathway Analysis software to identify affected target genes and their biological functions. PITX2（Paired-like homeodomain transcription factor 2） emerged as a common gene hit from separate screens targeting skeletal and muscular disorders, cell morphology, and tissue development. BBP decreased transcription of PITX2 and elevated expression of the microRNA miR-137, the predicted upstream negative regulator of PITX2. These data indicated that BBP affects PITX2 expression through miR-137 targeting of the 3' untranslated region of PITX2 mRNA. PITX2 down-regulation also decreased MyoD transcript levels in EN-MSCs. Our results demonstrate that BBP decreases EN-MSCs myogenic differentiation through up-regulation of miR-137, contribute to our understanding of EN-MSCs differentiation and underline the hazardous potential of environmental hormones.

並列關鍵字

AhR ； BBP ； granulosa cells ； endometrial mesenchymal stem cells ； miR-137 ； Phthalate ； PITX2

參考文獻

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4. Grande, S.W., et al., A dose-response study following in utero and lactational exposure to di(2-ethylhexyl)phthalate: effects on female rat reproductive development. Toxicol Sci, 2006. 91(1): p. 247-54.

5. Ma, M., et al., Exposure of prepubertal female rats to inhaled di(2-ethylhexyl)phthalate affects the onset of puberty and postpubertal reproductive functions. Toxicol Sci, 2006. 93(1): p. 164-71.

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