Development and validation of a method to deliver vitamin A to macrophages.
Development and validation of a method to deliver vitamin A to macrophages.
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DOI：
10.1016/bs.mie.2022.04.008
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发表时间：
2022
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巨噬细胞是动脉粥样硬化病变发展的关键参与者，它们促进局部和全身炎症。巨噬细胞在进入动脉壁时吞噬脂蛋白和细胞碎片，成为载脂泡沫细胞。虽然在泡沫细胞中发现的大多数脂质是甘油三酯和胆固醇，但这些细胞积累了几种具有生物活性的其他脂质，如维生素A和类胡萝卜素。维生素A在巨噬细胞和其他免疫细胞中具有很强的免疫调节作用。例如，巨噬细胞释放维生素A作为视黄酸来调节T细胞分化，但由于缺乏将维生素A加载到巨噬细胞中的适当实验模型，细胞内维生素A储存在该过程中的含义仍然难以捉摸。本研究的目的是开发一种可靠的方法来提供维生素A培养的小鼠巨噬细胞。我们的研究结果表明，巯基乙酸盐引起的腹腔巨噬细胞未能采取显着水平的维生素A时，提供的游离视黄醇。培养的巨噬细胞和腹腔中的巨噬细胞可以摄取视黄酯，视黄酯作为负载视黄酯的血清或视黄酯直接输注到腹腔中。在巨噬细胞裂解物的HPLC分析表明，腹腔注射方法的结果在四倍以上的维生素A负载效率比视黄酯加载血清添加到培养的细胞。这两种替代方法提供了用维生素A负载巨噬细胞的有效且可靠的方法，用于下游应用，例如研究细胞内维生素A的基因调控运输和巨噬细胞的维生素A释放。
Macrophages are critical players in the development of atherosclerotic lesions, where they promote local and systemic inflammation. Macrophages engulf lipoproteins and cell debris upon entry into the arterial wall, becoming lipid-laden foam cells. While most lipids found in foam cells are triglyceride and cholesterol, these cells accumulate several other lipids with bioactive properties, such as vitamin A and carotenoids. Vitamin A has strong immunomodulatory actions in macrophages and other immune cells. For example, macrophages release vitamin A as retinoic acid to modulate T cell differentiation, but the implication of intracellular vitamin A stores in this process remains elusive due to the lack of an adequate experimental model to load vitamin A into macrophages. The purpose of this study was to develop a reliable method to deliver vitamin A to cultured murine macrophages. Our results show that thioglycolate-elicited peritoneal macrophages fail to take up significant levels of vitamin A when provided as free retinol. Cultured macrophages and macrophages in the peritoneal cavity can take up retinyl esters, either as retinyl ester-loaded serum or retinyl esters infused directly into the peritoneal cavity. HPLC analyses in macrophage lysates revealed that the intraperitoneal injection method results in a fourfold greater vitamin A loading efficiency than retinyl ester-loaded serum added to cultured cells. These two alternative methods provide an efficient and reliable methodology to load macrophages with vitamin A for downstream applications such as studies of gene regulation trafficking of intracellular vitamin A, and vitamin A release from macrophages.