棒麴黴素對小鼠胚胎的凋亡及其體外發育的影響

Effect of Patulin on the Apoptosis and In-Vitro Embryonic Development of Mouse Blastocysts

詹家韋(Joseph Renz Marion Santiago Dela Cruz)

指導教授 ： 詹文雄

中原大學/理學院/生物科技研究所/碩士(2020年)

https://doi.org/10.6840/cycu202000299

摘要

棒麴黴素是一種由青黴屬、麴黴屬及絲衣黴屬等不同屬的菌種所分泌的黴菌毒素，並且主要由青黴菌和擴展青黴所產生。這種黴菌毒素主要存在於新鮮蘋果及其產品（蘋果汁、蘋果酒醋等）中。由於蘋果為很普及的食物，所以孕婦食用遭黴菌毒素污染的蘋果及其產品時，黴菌毒素對胎兒發育的影響則仍不清楚，因此探討棒麴黴素對胚胎發育的影響是很重要的。本研究旨在釐清棒麴黴素對小鼠早期胚胎發育的影響及其作用機轉。實驗以5-10 μM 的棒麴黴素與小鼠囊胚期胚胎共培養，透過 TUNEL 細胞凋亡分析及 Hoecsht 染色分析探討細胞凋亡和存活率，並以光學顯微鏡觀察5天內胚胎的體外發育型態。此外，以雙重染色法分析內細胞群或滋養層細胞數目與增生情形；而以兩種螢光染色來偵測兩種棒麴黴素所誘導的胚胎細胞凋亡機制，包括以DCF-DA染劑來分析活性氧族群的改變，而利用DiOC6染劑來偵測粒線體膜電位的變化。結果發現棒麴黴素於濃度大於5 μM時會誘導小鼠胚胎細胞凋亡並抑制其細胞增生，且會增加胚胎細胞內的氧化壓力和誘發粒線體膜電位的喪失而引發胚胎細胞凋亡，而棒麴黴素所導致的內細胞團細胞凋亡進而傷害胚胎的發育潛能。總括來說，本研究結果推測在懷孕期間攝入受棒麴黴素所污染的食物或食品時具有導致胚胎發育損傷進而造成流產的風險。

關鍵字

棒麴黴素 ； 細胞凋亡 ； 囊胚 ； 氧化壓力

並列摘要

Patulin is a mycotoxin produced by different genera of fungi such as Penicillium, Aspergillus, and Byssochlamys species and is primarily produced by Penicillium patulum and Penicillium expansum. This mycotoxin is mostly found in fresh apples and its products (Juice, Cider Vinegars, etc.). Due to apple’s popularity worldwide, there are some pregnant woman who initially consume this fruit and its products without knowing the possible effects of the unseen mycotoxin on the fetal development of their unborn child. Thus, the possible effects of Patulin on embryonic stages is important to observe. This study aims to investigate the different possible effects of Patulin in mouse early embryonic development and its mechanisms. For this purpose, mouse embryos (blastocyst) were treated with different concentration of Patulin ranging from 5 to 10 µM. Cellular viability and proliferation were evaluated by TUNEL and Hoecsht staining assay. Morphological development (outgrowth) of embryo in vitro was observed under light microscopy for 5 days for any visible changes. In addition, dual differential staining was used to identify whether the ICM or TE has undergone apoptosis. Two mechanisms of cellular apoptosis induced by Patulin were also detected by fluorescence staining namely: DCF-DA for reactive oxygen species (ROS) and DiOC6 for mitochondrial membrane potential (ΔΨm). The results revealed that >5 µM concentration of Patulin induces cellular apoptosis and inhibited cell proliferation of mouse embryo. ROS production and loss of mitochondrial membrane potential may cause apoptosis. Furthermore, Patulin could impair embryo’s developmental potential by inducing apoptosis in inner cell mass. Taken all together, it suggests that intake of Patulin could potentially harm the embryo during pregnancy leading to miscarriage.

並列關鍵字

Patulin ； Apoptosis ； Blastocyst ； Oxidative stress

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