脂蛋白相關基因在雞胚發育期間內胚層上皮細胞中的作用

Abstract

禽胚會依賴卵黃囊膜（Yolk sac membrane, YSM）吸收卵黃中的營養分，其中內胚層上皮細胞（Endodermal epithelial cells, EECs）為YSM直接接觸卵黃的細胞，負責吸收卵黃中營養分並運送至血液循環系統，提供發育中禽胚所需。過去的研究發現，EECs產出極低密度脂蛋白（Very low density lipoprotein, VLDL）的膽固醇酯（cholesteryl ester, CE）含量遠高於卵黃中VLDL因此推測膽固醇會在EECs中進行酯化，並重新包裝運送出EECs。然而，因為在EECs中檢測出載脂蛋白A1（Apolipoprotein A1, APOA1）的高表現量，我們認為EECs中高密度脂蛋白（High density lipoprotein, HDL）的生成和膽固醇酯的運輸至關重要。然而，目前文獻中尚未有EECs在HDL代謝中的直接研究。基於EECs的功能特性及其與雞胚肝臟的相似性，推測EECs可能如雞胚肝臟細胞透過內質網分泌途徑生成新生HDL，以此來運送胚胎發育所需的部分膽固醇。先前的研究證實Fatty acid binding protein 5 （FABP5）可增加VLDL出芽（budding）及脂肪酸生成，但FABP5在家禽EECs中的存在及其對VLDL和HDL代謝的影響尚未明確。因此本研究目的為釐清EECs中的脂質代謝模式，並通過增加FABP5的表現來調控脂蛋白的運輸效率以增加雞胚的脂質利用效率。 本研究分成兩階段，為驗證EECs中脂質的代謝模式，首先利用scRNA-seq分析孵化第4天（E4）及孵化第7天（E7）之單顆細胞，以找到EECs中影響脂蛋白運輸之關鍵機制。單細胞分析研究中，典型EECs群體（Cluster 2）中APOA1表現量排名第3，而VLDL的標誌基因APOB則僅排名63，顯示EECs可能偏向於產生更多APOA1相關的脂蛋白來運送脂質。分析孵化第18天之雞胚心臟及卵黃囊靜脈之血漿，發現其中卵黃囊膜靜脈血漿中HDL和LDL/VLDL的膽固醇濃度均顯著高於心臟血漿 （p < 0.05），而心臟 （78%）及卵黃囊靜脈 （69%）血漿中高比例的HDL濃度說明在雞胚孵化後期，HDL可能取代VLDL的功能，成為主要的膽固醇運輸載體。Cluster 2中高度表現AFP、ALDOB和TTR等與肝臟相關基因，同時也高度表現CDH17和EPCAM等腸道上皮之標誌基因，說明EECs同時具有肝臟與腸道的特性，展現了一種獨特的雙重功能性。為驗證調控FABP5表現是否影響脂蛋白運輸效率，我們利用EECs初代培養系統通過添加100 μM棕櫚酸、1500 μM油酸、30 nM Troglitazone和1400 μM離胺酸，探討營養分的添加對脂蛋白相關基因及三酸甘油酯（Triglycerides, TG）、總膽固醇（Total cholesterol, TC）和HDL膽固醇（HDLC）含量。基因表現分析顯示，在EECs中FABP5上調主要促進VLDL合成與運輸。Lysine處理會增加VLDL和HDL運輸並降低EECs中TC和HDLC，而TGZ處理會增加TG含量但可能限制VLDL組裝導致細胞中TG的累積。此外，FABP5啟動子截切分析顯示，移除PPARγ （-767 bp 至 -621 bp）和SREBF1（-621 bp 至 -373 bp）片段後，冷光表現呈上升趨勢，並發現可能的RE-1 silencing transcription factor （REST） 結合位點，暗示REST可能參與調控FABP5表達，此調控機制需進一步驗證。 綜上所述，EECs主要依賴HDL運輸脂質，而FABP5在EECs中的作用更側重於VLDL代謝，對HDL影響較小。本研究為雞胚脂質代謝提供了新見解，支持以HDL為主的運輸模式，未來可通過超高速離心驗證EECs中脂蛋白成分並探討如何調控這些脂質運輸蛋白的機制。

Avian embryo development relies on the yolk sac membrane （YSM） for nutrient absorption from the yolk, where endodermal epithelial cells （EECs） are located on the surface serve as the primary cells to for direct yolk access. These cells are responsible for absorbing yolk nutrients and transporting them into the circulation to support embryonic development. Previous studies have shown that EECs produce very-low-density lipoprotein （VLDL） with significantly higher cholesteryl ester （CE） content compared to yolk VLDL, suggesting that VLDL undergoes re-esterification and repackaging within EECs for lipid transport. However, the high expression of APOA1 in EECs, indicates that high-density lipoprotein （HDL） synthesis may be involved in cholesteryl ester transport. However, there are limited direct studies on HDL metabolism in EECs. Given the functional similarities between EECs and embryonic chick hepatocytes, it is hypothesized that EECs may generate nascent HDL through the endoplasmic reticulum secretory pathway, facilitating the transport of cholesterol essential for embryonic development. Previous studies have demonstrated that FABP5 enhances VLDL budding and fatty acid production, yet its presence and impact on VLDL and HDL metabolism in avian EECs remain unclear. Therefore, this study aims to investigate the lipoprotein profile in EECs and determine whether modulating FABP5 expression can enhance lipoprotein transport efficiency to improve lipid utilization in chick embryos. The study is divided into two parts. Initially, to validate the lipid metabolism patterns in EECs, single-cell RNA sequencing （scRNA-seq） was employed to isolate single cells from embryonic day 4 （E4） and day 7 （E7）, aiming to identify key mechanisms influencing lipoprotein transport. In the single-cell analysis, the typical EEC population （Cluster 2） exhibited APOA1 expression ranked third, while the VLDL marker gene APOB ranked only 63rd, suggesting that EECs may preferentially produce APOA1-associated lipoproteins for lipid transport. Subsequent analysis with the plasma from the embryonic heart and yolk sac vein at day 18 of incubation revealed significantly higher cholesterol concentrations of HDL and LDL/VLDL as compared to those from heart. Moreover, the high proportions of HDL in the plasma from heart （78%） and yolk sac vein （69%）indicate that HDL may surpass VLDL as the primary vehicle for lipid transport during late embryonic development. Cluster 2 also highly expressed liver-related genes such as AFP, ALDOB, and TTR, alongside intestinal epithelial markers CDH17 and EPCAM, suggesting that EECs possess dual characteristics of hepatocytes and intestinal epithelium without a clear bias toward either, reflecting a unique bifunctional role. To assess whether modulating FABP5 expression affects lipoprotein transport efficiency, the primary EEC culture system was utilized. Cells were treated with 100 μM palmitic acid, 1500 μM oleic acid, 30 nM troglitazone, and 1400 μM lysine to explore the impact of nutrient supplementation on lipoprotein-related gene expression and levels of triglycerides （TG）, total cholesterol （TC）, and HDL cholesterol （HDLC）in EECs. Gene expression analysis revealed that upregulation of FABP5 in EECs primarily enhances VLDL synthesis and transport. Lysine treatment increased VLDL and HDL transport while reducing TC and HDLC levels in EECs, whereas troglitazone treatment elevated TG content but potentially restricted VLDL assembly, leading to TG accumulation within cells. Additionally, FABP5 promoter deletion assays revealed reduced luciferase expression upon removal of PPARγ and SREBF1 regions, and identified a potential RE-1 silencing transcription factor （REST） binding site, suggesting REST may regulate FABP5 expression, though further validations are required. Collectively, these findings indicate that EECs primarily utilize HDL for cholesterol transport, while FABP5 in EECs is primarily associated with VLDL metabolism, with a limited influence on HDL. The study provides new insights into chick embryo lipid metabolism, supporting HDL as the primary vehicle for lipid delivery. In the future, ultracentrifugation can be used to validate the lipoprotein components in EECs and to investigate the mechanisms regulating these lipid transport proteins.