In vivo Liver Endocytosis Followed by Purification of Liver Cells by Liver Perfusion
In vivo Liver Endocytosis Followed by Purification of Liver Cells by Liver Perfusion
复制标题
DOI：
10.3791/3138
复制
发表时间：
2011-11-01
影响因子：
1.2
通讯作者：
Harris, Edward N.
中科院分区：
文献类型：
--
作者：
Gopalakrishnan, Sandhya;Harris, Edward N.
肝脏是哺乳动物身体的代谢中心，是血液的过滤器。肝脏的基本结构如图(1)所示，其中85%以上的肝脏肿块由肝细胞组成，其余15%的细胞团块由Kupffer细胞(KCs)、星状细胞(HSCs)和肝窦内皮细胞(SECs)组成。SEC形成肝脏内的血管壁，在细胞质中包含称为窗孔的特殊形态。细胞质开窗是指细胞内出现孔洞(类似于100微米)，因此SECs充当筛子，在其中大多数乳糜粒、乳糜粒残留物和大分子，而不是细胞，穿过肝细胞和HSC(图1)。由于缺乏基底膜，SECs和肝细胞之间的间隙形成了Disse空间。肝星状细胞占据这个空间，在调节和反应损伤、储存维甲酸和免疫调节肝脏方面发挥重要作用(2)。肝干细胞是体内最活跃的细胞之一，在其细胞表面显示一系列清道夫受体(3)。这些药物包括SR-A、Stablin-1和Stablin-2。一般情况下，小于230 nm的小胶体颗粒和缓冲相中的大分子被SECS摄取，而大颗粒和细胞碎片被KCs(4)吞噬。因此，从血液中大量清除细胞外物质，如糖胺多聚糖，在很大程度上取决于Secs的健康和内吞功能(5，6)。例如，血中透明质酸水平的升高预示着从轻度到重度的各种肝病(7)。除了一份报告(8)，没有永生化的SEC细胞系存在。即使是这种永生化的细胞系也是去分化的，因为它不表达初级SEC上存在的清道夫受体(我们的数据，未显示)。所有细胞生物学研究都必须在新鲜从动物身上获得的原代细胞上进行。不幸的是，在标准的培养条件下，SEC会去分化，必须在与动物隔离后的1到2天内使用。SECs的分化以Stabilin-2或HARE受体(9)、CD31的表达和胞浆窗口的存在为标志(1)。通过在培养液中添加血管内皮生长因子或在肝细胞条件培养液(10，11)中培养细胞，可以延长SECs的分化。在这篇报告中，我们将使用放射性标记的透明质酸肝素来证明SECs在完整器官中的内吞活性。然后，我们将从灌流的肝脏中纯化肝细胞和SECs来测量内吞作用。
The liver is the metabolic center of the mammalian body and serves as a filter for the blood. The basic architecture of the liver is illustrated in figure (1) in which more than 85% of the liver mass is composed of hepatocytes and the remaining 15% of the cellular mass is composed of Kupffer cells (KCs), stellate cells (HSCs), and sinusoidal endothelial cells (SECs). SECs form the blood vessel walls within the liver and contain specialized morphology called fenestrae within in the cytoplasm. Fenestration of the cytoplasm is the appearance of holes (similar to 100 mu m) within the cells so that the SECs act as a sieve in which most chylomicrons, chylomicron remnants and macromolecules, but not cells, pass through to the hepatocytes and HSCs (1) (Fig. 1). Due to the lack of a basement membrane, the gap between the SECs and hepatocytes form the Space of Disse. HSCs occupy this space and play a prominent role in regulation and response to injury, storage of retinoic acid and immunoregulation of the liver (2).SECs are among the most endocytically active cells of the body displaying an array of scavenger receptors on their cell surface (3). These include SR-A, Stabilin-1 and Stabilin-2. Generally, small colloidal particles less than 230 nm and macromolecules in buffer phase are taken up by SECs, whereas, large particles and cellular debris is endocytosed (phagocytosed) by KCs (4). Thus, the bulk clearance of extracellular material such as the glycosaminoglycans from blood is largely dependent on the health and endocytic functions of SECs (5,6). For example, an increase in blood hyaluronan levels is indicative of liver disease ranging from mild to more severe forms (7).With the exception of one report (8), there are no immortalized SEC cell lines in existence. Even this immortalized cell line is de-differentiated in that it does not express scavenger receptors that are present on primary SECs (our data, not shown). All cell biological studies must be performed on primary cells obtained freshly from the animal. Unfortunately, SECs dedifferentiate under standard culture conditions and must be used within 1 or 2 days upon isolation from the animal. Differentiation of SECs is marked by the expression of Stabilin-2 or HARE receptor (9), CD31, and the presence of cytoplasmic fenestration (1). Differentiation of SECs can be extended by the addition of VEGF in culture media or by culturing cells in hepatocyte conditioned medium (10,11).In this report, we will demonstrate the endocytic activity of SECs in the intact organ using radio-labeled heparin for hyaluronan for the SEC-specific Stabilin-2 receptor. We will then purify hepatocytes and SECs from the perfused liver to measure endocytosis.