鄰苯二甲酸甲苯基丁酯經由Mir-137調控PITX2降低人類子宮內膜間質幹細胞之分化能力

Benzyl butyl phthalate decreases the differentiation ability of endometrial mesenchymal stem cells through PITX2 regulated by Mir-137

張育嘉(Yu-Chia Chang)

指導教授 ： 蔡英美

高雄醫學大學/醫學院/醫學研究所碩士班/碩士(2015年)

摘要

近來台灣由於食安問題頻傳,使得環境荷爾蒙對人體健康影響的議題 更加受到重視。鄰苯二甲酸酯(phthalate esters)俗稱塑化劑為一環境荷爾 蒙,常被廣泛使用於許多加工產品,例如:玩具、化妝品、包裝食物的容 器和醫療器具等。間質幹細胞(mesenchymal stem cells, MSCs)為一多能 的間質細胞,具有分化成脂肪、硬骨、軟骨和肌肉細胞的能力。而且間質 幹細胞可以從脂肪組織、臍帶和子宮內膜等成體組織中分離出來。目前 BBP 對於間質幹細胞分化能力的影響依然不甚清楚。因此我們想要探討 BBP 影響間質幹細胞分化過程中所扮演的角色以及調控的路徑。我們經由 細胞培養、細胞化學染色和 real-time qPCR 等方法發現 BBP 會降低間質幹 細胞分化能力的現象,所以我們使用了 cDNA microarray assay 和 Ingenuity Pathways Analysis (IPA) software 找出在間質幹細胞上 BBP 的目標基因以 及其所影響的相關路徑,而我們找到並經由實驗證明 BBP 會降低 PITX2 的表現而降低間質幹細胞分化成肌肉細胞的能力。更進一步的我們使用了 microRNA 的預測軟體 miRanda 找出了 PITX2 的可能上游調控者:miR-137。 接著我們也由細胞實驗證實 BBP 會經由 miR-137 降低 PITX2 的表現而降 低 EN-MSCs 分化成肌肉的能力。我們也經由動物實驗證明 BBP 會降低 EN-MSCs 分化成肌肉的能力。因此由我們的實驗結果可知環境荷爾蒙 (BBP)對於人類間質幹細胞的分化以及組織發育具有潛在性的危害。

關鍵字

塑化劑 ； 鄰苯二甲酸甲苯基丁酯 ； 人類子宮內膜間質幹細胞 ； microRNA ； PITX2

並列摘要

Recently, the safety issues of food have become much more important in Taiwan, and the environmental hormones are the most important factors about that. The environmental hormones, phthalate esters, are widely used for a lot of products, such as toys, cosmetics, food packaging materials, medical devices, and so on. Mesenchymal stem cells (MSCs) are multipotent stromal cells, which can differentiate into adipogenic, osteogenic, chondrogenic and myogenic lineages. MSCs are easily isolated from various adult tissues, such as adipose tissue, umbilical cord, and endometrium. The correlation of BBP and the differentiation ability of MSCs is still unknown. Therefore, we investigate the effect of BBP on differentiation ability and find the novel signaling pathway in endometrial mesenchymal stem cells (EN-MSCs).
EN-MSCs have the ability to differentiate into adipogenic, osteogenic, chondrogenic and myogenic lineages. In this study, we found that BBP decreased the differentiation ability of osteogenesis and myogenesis in EN-MSCs. To further examine the role of BBP in EN-MSCs, we used the cDNA microarray assay to screen BBP regulation of gene expression and analyzed the biological function network with Ingenuity Pathways Analysis (IPA) software in EN-MSCs. We demonstrated that the candidate genes, SRC and PITX2 regulate concomitantly the Skeletal and Muscular Disorders, the Cell Morphology, Tissue Development from the Top Bio Functions group. We further used a web-based microRNA target prediction program (miRanda) to search for potential targets. We confirmed the significant targets with real-time qPCR by overexpression of microRNA. Thus we explored the novel signaling pathway that miR-137 can target PITX2 when BBP treatment in EN-MSCs. Then we validated the in vivo results by immunohistochemistry stain in the tissue specimen obtained from the animal model. Finally, we confirmed that BBP reduced the differentiation ability of myogenesis of EN-MSCs in muscle regeneration animal model.
Our study shows that BBP decreases the differentiation ability of EN-MSCs through activation of miR-137 expression. Subsequently, miR-137 targets PITX2 to affect myogenesis. These findings contribute to our understanding of the differentiation ability of EN-MSCs in human, and the hazard potential of environmental hormone.

並列關鍵字

phthalate ； benzyl butyl phthalate ； endometrial mesenchymal stem cells ； microRNA ； PITX2

參考文獻

台灣行政院環境保護署（http://www.epa.gov.tw/）

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