丙酮醛對小鼠早期胚胎發育與DNA甲基化之影響

Effect of methylglyoxal on development and DNA methylation in mouse early stage embryo

高儷珍(Li-Zhen Gao)

指導教授 ： 詹文雄

中原大學/理學院/生物科技研究所/碩士(2018年)

https://doi.org/10.6840/cycu201800873

摘要

甲基乙二醛(methylglyoxal；MG)又稱為丙酮醛或2-氧丙醛，是一種糖分解產物，對細胞具有毒性及抗病毒和抑制腫瘤的效果。在日常飲食中能由高果糖玉米糖漿飲料和加工品及高溫烹飪，尤其是炸、煎、和烤食物中攝取的到。有研究指出丙酮醛是葡萄糖的代謝產物會進行醣化 (glycation)反應與蛋白質結合形成不可逆的糖化终產物 (advanced glycation end products, AGEs)，進一步產生活性氧(ROS)和自由基 (Free Radical)，導致細胞毒性和細胞凋亡。在糖尿病患者高血糖狀態下在各個組織中會增加葡萄糖代謝進而使血液中丙酮醛增加，這對本身有糖尿病的婦女或懷孕期間產生妊娠糖尿病者會對胚胎及母體造成影響。但丙酮醛對胚胎二細胞時期到囊胚期發育期間基因表達的影響目前尚不清楚。因此本研究利用小鼠二細胞時期胚胎與丙酮醛共培養，探討丙酮醛對小鼠胚胎發育及 DNA 甲基化之影響。將小鼠早期胚胎與50 M丙酮醛共培養 72 小時後，發現胚胎發育率有顯著下降，並且發育到囊胚時期的胚胎細胞數目顯著減少。再進一步以5-甲基胞嘧啶免疫螢光染色和重亞硫酸氫鹽定序方式分析丙酮醛對胚胎發育DNA甲基化之影響，結果顯示丙酮醛共培養的胚胎中GATA6、BMP4 基因與控制組相比有顯著差異而SYP基因在各組之間並沒有差異。這些結果證實丙酮醛對早期胚胎發育具有傷害效應，並透過影響早期胚胎的基因甲基化程度而影響胚胎的發育。

關鍵字

丙酮醛 ； 早期胚胎 ； DNA甲基化

並列摘要

Methylglyoxal ( MG ) also called as pyruvic aldehyde or 2-oxopropanal is a highly reactive dicarbonyl compound which is inevitably formed as a by-product of glycolysis. The previous studies have shown that MG has a cytotoxic effect in vitro and in vivo. Additionally, the anti-virus and inhibition of tumorigenesis effects of MG were also found. Further investigations have suggested that MG could be obtain through intake of high-fructose corn syrup beverages, processed food products, and foods which undergone high-temperature cooking. When MG reacts with amino acid it can induce reactive oxygen Species (ROS) and free radical leading to subsequent cytotoxicity and apoptosis. In hyperglycemia state of diabetic patients, the formation of the glucose metabolite pyruvic aldehyde can be increased in various tissues and women who are diagnosed with gestational diabetes during pregnancy should pay more attention to their condition for it will affect the fetus development. Previous studies have demonstrated the cytotoxic effects of MG on mouse oocyte maturation, fertilization, blastocyst development, and a further association with defects in subsequent embryonic development. In this study, we further examined 2-cell stage embryos of mice, co-incubated with MG to investigate whether MG has an effect on embryos development and DNA methylation. The co-cultured of early embryos with 50 M of MG for 72 hours has a significant decrease on embryonic development and the number of embryonic cells that developed to the blastocyst stage was significantly reduced. Moreover, the effects of MG on DNA methylation for embryo development were analyzed by 5-methylcytosine immunofluorescence staining and bisulfite sequencing PCR. The results showed that the methylation levels of the GATA6 and BMP4 genes were different from the control group and SYP gene has no difference in various MG-treated groups. These study results clearly demonstrated that MG has impairments on early stage embryonic development via influence of DNA methylation.

並列關鍵字

Methylglyoxal ； early stage embryos ； DNA methylation

參考文獻

1. Allen, R.E., Lo, T.W., and Thornalley, P.J. (1993). Inhibitors of glyoxalase I: design, synthesis, inhibitory characteristics and biological evaluation. Biochem Soc Trans 21, 535-540.

2. Artus, J., and Chazaud, C. (2014). A close look at the mammalian blastocyst: epiblast and primitive endoderm formation. Cell Mol Life Sci 71, 3327-3338.

3. Benvenisty, N., Mencher, D., Meyuhas, O., Razin, A., and Reshef, L. (1985). Sequential changes in DNA methylation patterns of the rat phosphoenolpyruvate carboxykinase gene during development. Proc Natl Acad Sci U S A 82, 267-271.

4. Berezney, R., and Jeon, K.W. (1996). International Review of Cytology: Volume 162A (Elsevier Science).

5. Bergsmedh, A., Donohoe, M.E., Hughes, R.A., and Hadjantonakis, A.K. (2011). Understanding the molecular circuitry of cell lineage specification in the early mouse embryo. Genes (Basel) 2, 420-448.