母親處理DNA甲基轉移酶抑制劑對ApoE-/-小鼠子代動脈粥狀硬化發展和脂質代謝之研究

The study of maternal DNA methyltransferase inhibitor treatment on the development of atherosclerosis and lipid metabolism in adult Apolipoprotein E deficient mice offspring

洪廷芸(Ting-Yun Hung)

指導教授 ： 林甫容

國立臺灣大學/生命科學院/生化科技學系/碩士(2020年)

https://doi.org/10.6342/NTU202003815

摘要

發展規劃 (Developmental programming) 指的是胎兒和新生兒暴露的環境將對成年後的生理功能和疾病風險產生深遠的影響。過去的研究指出，孕婦的營養過剩會造成子代的心血管和代謝方面的問題，其中可能的機制是透過表觀遺傳學。我們實驗室先前研究發現，懷孕前後誘發高膽固醇血症的 ApoE-/- 母鼠所生出的子代在成年後較容易罹患非酒精性脂肪肝和動脈粥狀硬化，且基因表現改變與DNA甲基化程度相關。故本研究的假說為：親代母鼠施打DNA 甲基轉移酶抑制劑 5-aza-2′-deoxycytidine (5Aza-dC) 可以藉由調控DNA甲基化的程度而減緩子代動脈粥狀硬化的發展。
我們使用 ApoE -/- 母鼠餵食控制組飲食 (Control diet, CD)，並在懷孕前以及哺乳期間施打 PBS 或 5Aza-dC，親代母鼠共有二組：CD + PBS 以及 CD + 5Aza-dC。所產出的子代在離乳後統一餵食 WD (分別為PBS + CW、5Aza-dC + CW)。結果發現在 16 週齡，5Aza-dC + CW 組子代雄鼠的動脈粥狀硬化程度與 PBS + CW 組相比顯著減緩；5Aza-dC + CW 組子代雌鼠與 PBS + CW 組相比下，動脈粥狀硬化也有減緩的趨勢。且伴隨著雄和雌鼠的脾臟中CD4+ T細胞比例、雄鼠脾臟中CD8+ T 細胞和以及雌鼠淋巴結中促發炎 Th1 細胞的比例與PBS + CW組相比顯著下降。另外， 5Aza-dC + CW 組與 PBS + CW 組雄鼠相比，血清及肝臟三酸甘油酯的累積均較低，但是並沒有改變參與肝臟脂質形成、氧化及運送的轉錄因子及相關基因的 mRNA 表現量。因此，我們推測親代母鼠施打 5Aza-dC 可能使子代的脂質吸收能力下降進而減緩肝臟脂質堆積。為了印證此假說，第二個部分的實驗我們使用ApoE +/- 公鼠餵食 WD，探討直接施打 5Aza-dC 是否會影響小鼠對於脂質的吸收。實驗結果顯示，在攝食量相同的情形下，施打5Aza-dC公鼠糞便排出的三酸甘油酯含量上升。且與施打 PBS 組相比，施打5Aza-dC公鼠在餐後的三酸甘油酯吸收及小腸乳糜微粒的分泌皆顯著下降。探討其可能分子機制，我們發現施打5Aza-dC會顯著下調公鼠空腸中的 Slc27a4 (FATP4) 以及 Mttp 的 mRNA 表現量，並可能因此降低小腸乳糜微粒的分泌。
綜合以上研究可知，親代母鼠施打 5Aza-dC 可能藉由改變脾臟 CD8+ T 細胞、CD4+ T 細胞及淋巴結 Th1 細胞的比例以減緩子代的動脈粥狀硬化；另外，親代母鼠施打 5Aza-dC 可能藉由降低脂質的吸收而減少子代肝臟的三酸甘油酯堆積。此假說也由施打 5Aza-dC的 ApoE +/- 公鼠有降低小腸的乳糜微粒分泌及減少三酸甘油酯吸收得到印證。後續的研究會著重在 5Aza-dC 是否會透過母體效應影響子代脂質的吸收能力以及探討其中的機制。

關鍵字

發展規劃 ； DNA甲基轉移酶抑制劑 ； 動脈粥狀硬化 ； T細胞

並列摘要

The environment encountered in fetal and neonatal life exerts a profound influence on physiological function and risk of disease in adult life. A number of epidemiologic and animal studies indicated that maternal over-nutrition during pregnancy leads to the development of cardiovascular and metabolic disease in the adult offspring likely through alterations in DNA methylation. However, the mechanisms underlying the fetal programming of adult disease are still unclear. We aim to investigate whether the maternal treatment of DNA methyltransferase inhibitor (5-aza-2′-deoxycytidine, 5Aza-dC) could ameliorate the development of atherosclerosis in the offspring.
In our study, ApoE -/- dams were fed with a control diet (CD), and 5Aza-dC was administrated before pregnancy and during lactation. All offspring were fed with a WD after weaning. Maternal 5Aza-dC treatment ameliorated the development of atherosclerosis in offspring likely through decreasing the CD8+ T cell and CD4+ T cell population of spleen and Th1 cell population in lymph nodes. Furthermore, we showed that upon maternal CD feeding, 5Aza-dC administration decreased hepatic triglyceride accumulation without affecting hepatic de novo lipogenesis, lipid oxidation and lipid transport in male offspring. To examine if 5Aza-dC treatment directly alters lipid metabolism, WD-fed ApoE+/- mice were treated with 5Aza-dC for 6 weeks. Compared to PBS-treated group, 5Aza-dC administration remarkably lowered triglyceride secretion from intestine to circulation in response to an acute dietary fat challenge. In addition, fecal triglyceride levels were significantly higher in 5Aza-dC-treated group compared with those in PBS-treated group. We also found that 5Aza-dC treatment significantly decreased Slc27a4 (FATP4) and Mttp mRNA expression in jejunum of the mice.
Taken together, maternal 5Aza-dC treatment may ameliorate the atherosclerosis through altering lymphocyte population in spleen and lymph nodes of adult offspring. Furthermore, maternal 5Aza-dC treatment may reduce hepatic triglyceride levels through reducing lipid absorption in offspring. Future research will be focused on whether and how triglyceride absorption is affected by maternal 5Aza-dC treatment.

並列關鍵字

Fetal programming ； 5-aza-2′-deoxycytidine ； atherosclerosis ； T cell

參考文獻

陳信宇 (2018). ApoE基因缺乏母鼠高膽固醇血症促進子代動脈粥狀硬化進程之機制研究. In 生化科技學系 (臺灣大學), pp. 1-137.

黃巧虹 (2020). 初探母體5-aza對子代動脈粥狀硬化病程之影響. In 生化科技學系 (臺灣大學), pp. 1-143.

李依靜 (2020). ApoE基因缺乏母鼠施打DNA甲基轉移酶抑制劑對子代小鼠動脈粥狀硬化進程之影響：巨噬細胞極化分化之角色. In 生化科技學系 (臺灣大學), pp. 1-195.

Ait-Oufella, H., Herbin, O., Bouaziz, J.-D., Binder, C. J., Uyttenhove, C., Laurans, L., Taleb, S., Van Vré, E., Esposito, B., Vilar, J., Sirvent, J., Van Snick, J., Tedgui, A., Tedder, T. F., Mallat, Z. (2010). B cell depletion reduces the development of atherosclerosis in mice. The Journal of experimental medicine 207(8), 1579-1587.

Ait-Oufella, H., Salomon, B. L., Potteaux, S., Robertson, A.-K. L., Gourdy, P., Zoll, J., Merval, R., Esposito, B., Cohen, J. L., Fisson, S., Flavell, R. A., Hansson, G. K., Klatzmann, D., Tedgui, A., Mallat, Z. (2006). Natural regulatory T cells control the development of atherosclerosis in mice. Nature Medicine 12(2), 178-180.