Ganesan Latha P, Mates Jessica M, Cheplowitz Alana M, Avila Christina L, Zimmerer Jason M, Yao Zhili, Maiseyeu Andrei, Rajaram Murugesan V S, Robinson John M, Anderson Clark L
Department of Internal Medicine, The Ohio State University, Columbus, OH 43210, United States.
Department of Surgery, The Ohio State University, Columbus, OH 43210, United States.
Sci Rep. 2016 Feb 11;6:20646. doi: 10.1038/srep20646.
Cholesterol from peripheral tissue, carried by HDL, is metabolized in the liver after uptake by the HDL receptor, SR-B1. Hepatocytes have long been considered the only liver cells expressing SR-B1; however, in this study we describe two disparate immunofluorescence (IF) experiments that suggest otherwise. Using high-resolution confocal microscopy employing ultrathin (120 nm) sections of mouse liver, improving z-axis resolution, we identified the liver sinusoidal endothelial cells (LSEC), marked by FcγRIIb, as the cell within the liver expressing abundant SR-B1. In contrast, the hepatocyte, identified with β-catenin, expressed considerably weaker levels, although optical resolution of SR-B1 was inadequate. Thus, we moved to a different IF strategy, first separating dissociated liver cells by gradient centrifugation into two portions, hepatocytes (parenchymal cells) and LSEC (non-parenchymal cells). Characterizing both portions for the cellular expression of SR-B1 by flow cytometry, we found that LSEC expressed considerable amounts of SR-B1 while in hepatocytes SR-B1 expression was barely perceptible. Assessing mRNA of SR-B1 by real time PCR we found messenger expression in LSEC to be about 5 times higher than in hepatocytes.
由高密度脂蛋白（HDL）携带的外周组织胆固醇，在被HDL受体SR-B1摄取后于肝脏中代谢。长期以来，肝细胞一直被认为是肝脏中唯一表达SR-B1的细胞；然而，在本研究中，我们描述了两个不同的免疫荧光（IF）实验，结果却并非如此。我们使用高分辨率共聚焦显微镜，采用小鼠肝脏的超薄（120nm）切片，提高了z轴分辨率，鉴定出以FcγRIIb为标记的肝窦内皮细胞（LSEC）是肝脏中表达大量SR-B1的细胞。相比之下，用β-连环蛋白鉴定的肝细胞表达水平要低得多，尽管对SR-B1的光学分辨率不足。因此，我们采用了另一种IF策略，首先通过梯度离心将解离的肝细胞分为两部分，即肝细胞（实质细胞）和LSEC（非实质细胞）。通过流式细胞术对这两部分细胞的SR-B1细胞表达进行表征，我们发现LSEC表达大量的SR-B1，而肝细胞中SR-B1的表达几乎难以察觉。通过实时PCR评估SR-B1的mRNA，我们发现LSEC中的信使表达比肝细胞中的高约5倍。
