發動蛋白於日本鵪鶉卵黃囊膜之內胚層上皮細胞扮演協助脂質吸收之重要角色

Abstract

禽胚孵化之中後期為其生長發育的重要階段，主要仰賴腹腔延伸出之卵黃囊膜（Yolk sac membrane, YSM）將卵黃包覆，由內胚層上皮細胞（Endodermal epithelial cells, EECs）對脂質進行吸收及利用，然而YSM對脂質吸收及利用的具體機制仍尚待釐清，本研究將探討EECs對脂質的吸收機制，期望期望研究結果對家禽產業有所貢獻。卵黃內大部分的脂質為由包覆三酸甘油酯的極低密度脂蛋白（Very low density lipoprotein, VLDL）所組成，而VLDL主要透過網格蛋白媒介包吞作用（Clathrin-mediated endocytosis, CME）進入細胞。CME仰賴許多蛋白共同協調，其中發動蛋白（Dynamin, DNM）之功能較為獨立且易於調控，因此本研究將主要探討DNM是否於EECs中扮演協助脂質吸收的角色，以了解EECs利用脂質機制與後續對胚發育影響之研究。 本研究以日本鵪鶉作為試驗動物，首先探討孵化期間YSM中乙醯-輔酶A-膽固醇乙醯轉移酶（Sterol O-acyltransferase, SOAT）、apolipoprotein B（ApoB）、cell death-inducing DFFA-like effector（CIDE），以及DNM之mRNA表現，希望建立出YSM對脂質吸收、修飾和脂蛋白組成之路徑圖，再從中判定DNM於YSM的重要性。結果發現YSM之DNM1 mRNA於入孵第10天時有最高表現，SOAT1主要表現於入孵第10-14天，CIDEA及apoB則分別於入孵第16及18天有最高表現量，並皆與入孵第2天有顯著差異，由此結果我們認為DNM1於YSM中可能確實扮演協助脂質吸收的角色。其作用促使脂質進入YSM後經SOAT1進行膽固醇轉酯，並由apoB及CIDEA重新合成VLDL後再運送禽胚體內提供生長所需能量。接著本試驗將直接於入孵第3天之日本鵪鶉胚卵黃施打DNM之抑制藥物：dynasore，並於孵化第10天探討其發育狀況，以分析DNM於YSM之重要性。其結果發現相較於對照組、操作對照對照組以及載體對照組（DMSO），dynasore處理確實會影響胚的發育。接著由EECs層面嘗試對其DNM進行抑制及活化，並分析EECs對螢光標定VLDL的吸收是否會受到影響。Dynasore對EECs DNM進行抑制的結果發現，經過dynasore處理20及30分鐘，其細胞內的螢光訊號顯著低於未受dynasore處理之對照組。試驗進一步分析EECs對不同濃度VLDL之抑制效果，結果顯示不論800、1050及1300 µg/mL 之VLDL，經dynasore處理30分鐘皆能有效抑制EECs對其進行吸收。接著我們推測EECs若缺乏脂質，其細胞內DNM1、SOAT1、apoB及CIDEA的mRNA 表現也會受到影響，試驗中以dynasore處理EECs 3小時，抑制其對VLDL的吸收長達1天再萃取其RNA，然而mRNA之結果發現經過dynasore處理其基因表現皆不會受到改變。最後試驗利用S-Nitroso-L-glutathione（GSNO）對DNM進行活化，結果發現將EECs處理GSNO 30分鐘，再與800 µg/mL之螢光標定VLDL培養15及20分鐘，EECs內的螢光訊號會顯著的高於其對照組。 綜合上述，我們發現DNM確實於YSM中扮演協助脂質吸收的重要角色，此外也成功對EECs之DNM進行調控。然而，本試驗DNM1、SOAT1、apoB及CIDEA的mRNA表現並不會因為EECs內脂質的減少而受到改變，建議未來使用其他方法探討其脂質利用相關蛋白活性。儘管如此，希望未來能利用此研究結果，進一步探討EECs對脂質的利用機制，並期望最終能改善禽胚之發育狀況。

During avian embryonic development mid and late stages are critical, of which the yolk sac membrane (YSM) extends from the avian abdomen to embrace the whole yolk, and endodermal epithelial cells (EECs) within the YSM are liable for the absorption and utilization of the lipids. However, the mechanism of lipids absorption and utilization by YSM are still unclear. In this study, we investigated lipid absorption mechanism in EECs, in attempt to make contributions to poultry industry. The lipids in the yolk are mostly composed of the very low density lipoprotein (VLDL), whose uptake mainly depends on the clathrin-mediated endocytosis (CME) on the cell membranes, CME relies on the coordination of lot of proteins to help membrane invagination, and the vesicle formation through the regulation of dynamin (DNM). In addition, we focused on the role of DNM in the mechanism of lipids utilization in EECs. We utilized Japanese quails as an animal model, to examine mRNA expression patterns of sterol O-acyltransferase 1 (SOAT1), apolipoprotein B (ApoB), families of cell death-inducing DFFA-like effector (CIDE) and DNM in YSM along the development and to establish the pathway of lipids absorption, modification and lipoprotein assembly for the regulation of DNM in YSM. The mRNA expression of DNM1 reached a peak at day 10 of incubation, SOAT1, CIDEA and ApoB expression were expressed aboundently during incubation day 10-14,16 and 18 respectively, and all the results were significantly different with those at day 2 (P < 0.05), suggesting DNM1 helps the lipids absorption in YSM. Next, a DNM inhibitor: dynasore was injected into fertilized eggs of Japanese quail at incubation day 3, and the phenotypes were observed at day 10. Results demonstrated the complete development percentage of embryos were significantly lower in the dynasore group than the controls, sham and vehicle-groups (P<0.05). The activities of DNMs were also verified by the absorptions of fluorescence labeled-VLDL (DiI-yVLDL) in EECs. Fluorescent signals in EECs were decreased significantly after pre-treatments of dynasore for 20 and 30 minutes. The absorptions of VLDL at various levels (800, 1050, and 1300 µg/mL of DiI-yVLDL) were blocked in EECs with the pre-treated dynasore. Third, we examine if the mRNA levels of DNM1, SOAT1, ApoB and CIDEA were affected in EECs, due to the lack of lipid resources. EECs were pre-treated with dynasore for 3 hours to inhibit the VLDL absorption for 24 hours before the mRNA extraction. Nonetheless, all these gene expression levels were not altered. Finally, we tested treating EECs with S-Nitroso-L-glutathione (GSNO, a DNM activator) for 30 minutes, and results showed that the intake of DiI-yVLDL was increased significantly by GSNO. In conclusion, DNM indeed serves a critical role of the lipids absorption in YSM. Furthermore, we successfully manipulated the DNM activity in EECsm despite that the mRNA expression of DNM, SOAT1, ApoB and CIDEA were not changed in EEC, suggesting the amount and activities of these target gene should be analyzed by other methods. These results delineated the mechanism of lipids utilization in EECs, and way improve the avian embryonic development in the future.