Marginating肝白细胞的选择性收获
Summary
Marginating-hepatic leukocytes exhibit unique characteristics and distinct immunological functions compared to other leukocyte populations. Here we describe a method for selective harvesting of this specific hepatic cell population, through forced perfusion of the liver of rats or mice. Marginating-hepatic leukocytes seem critical in determining susceptibility to hepatic-related diseases and metastases.
Abstract
Marginating-hepatic (MH) leukocytes (leukocytes adhering to the sinusoids of the liver), were shown to exhibit unique composition and characteristics compared to leukocytes of other immune compartments. Specifically, evidence suggests a distinct pro- and anti-inflammatory profile of the MH-leukocyte population and higher cytotoxicity of liver-specific NK cells (namely, pit cells) compared to circulating or splenic immunocytes in both mice and rats. The method presented herein enables selective harvesting of MH leukocytes by forced perfusion of the liver in mice and rats. In contrast to other methods used to extract liver-leukocytes, including tissue grinding and biological degradation, this method exclusively yields leukocytes from the liver sinusoids, uncontaminated by cells from other liver compartments. In addition, the perfusion technique better preserves the integrity and the physiological milieu of MH leukocytes, sparing known physiological responses to tissue processing. As many circulating malignant cells and infected cells are detained while passing through the liver sinusoids, physically interacting with endothelial cells and resident leukocytes, the unique MH leukocyte population is strategically located to interact, identify, and react towards aberrant circulating cells. Thus, selective harvesting of MH-leukocytes and their study under various conditions may advance our understanding of the biological and clinical significance of MH leukocytes, specifically with respect to circulating aberrant cells and liver-related diseases and cancer metastases.
Introduction
在肝血窦包含对生物体关键的各种免疫活动，众多的白细胞亚型。例如，marginating肝（MH +）自然杀伤（NK）细胞，也被称为坑的细胞，特征在于形态为大颗粒淋巴细胞（LGLs）和功能作为自发性细胞毒性的能力的白细胞，使肝-抵抗建立血 – 源性肿瘤转移。本文提出的方法的目标是使氢白细胞选择性收割，为了研究这个重要和独特的细胞群体（和免疫隔室），并阐明各种操作对这些特定细胞的影响（ 例如 ，免疫激活）。
尽管免疫的故障消除显影原发性肿瘤，在癌症患者和动物模型的证据表明免疫系统可控制循环肿瘤细胞，微转移，和残留病THROUGH细胞介导的免疫（CMI）。然而， 在体内的功能和在人的体外研究和在动物中，这表明，大多数的自体肿瘤细胞是通过循环或脾白细胞1,2-细胞毒性抗这些之间的明显不一致。此不一致可能归因，至少部分地明显的白细胞亚群，即marginating肝（正弦）白细胞和其活化NK细胞，即坑细胞3的亚群在体内存在。的确，从我们的实验室未发表的数据表明，在F344大鼠的同基因肿瘤细胞（MADB106），其被发现循环和脾白细胞抗性，通过MH-NK细胞4裂解。因此，是所谓的“NK抗性”，以循环白细胞的肿瘤细胞可以通过MH-NK细胞来控制。值得注意的是，在小鼠中只有下列体内免疫MH-NK的增强的活性是明显的刺激（ 例如，通过使用保利我的：C或CpG的-C）5。
肝特异性NK细胞（MH-NK）位于正弦管腔内，附着在血管内皮细胞和枯否氏细胞。 MH-NK细胞是专门特征在于球形致密颗粒和杆芯囊泡6，其含有酸性磷酸酶如溶酶体酶，和穿孔和粒酶作为生物活性物质7,8。与循环NK细胞相比，MH-NK细胞表现出的颗粒和囊泡9-11的较高数目和大小。下炎性病症，被示出的MH-NK细胞表现出的LFA-1 12的更高的表达相比，循环NK细胞。这种增强的表达可能构成了一个机制，MH-NK细胞是针对某些肿瘤细胞比循环NK细胞13,14多种细胞毒性。 nterestingly， 下列体外孵育白细胞介素（IL）-2，MH-NK细胞变大，并它们的数量和颗粒的尺寸增加，所有这些都与一个亲音响淋巴因子激活的杀伤的勒细胞（LAK）15相一致。
活性氢白细胞不能通过的标准肝白细胞采收方法，其基于研磨和组织的生物降解独占获得。相比于标准的方法本文描述的我们的灌注方法有两个主要优点。首先，灌注的方法选择性地收割MH白细胞，防止污染由其他肝病车厢其他白细胞。其次，灌注技术更好保持完整性，活性，和MH白细胞的生理环境，不像组织处理方法损害细胞或改变它们的形态，和由于组织损伤，诱导各种因素的释放是显着调节免疫活动。
肝脏是癌转移所涉及的主要靶器官第二对各种感染16。当MH细胞表现出独特的特点是，研究在各种条件下该特定人群相对于这些疾病是很重要的。例如，它是值得注意的各种生物反应调节剂是全身性免疫激活（ 如聚I：C或CpG的-C）已经显示激活比循环白细胞5 MH-白细胞多。
Protocol
Representative Results
Discussion
本文提出的肝脏灌注方法使选择性采伐和marginating肝白细胞的独特人口的研究。肝NK细胞，也被称为坑细胞3，构成驻留在肝窦鲜明的NK细胞群。它们在大鼠，小鼠17和在人类18,19找到。相比孤立外周血NK细胞，坑的细胞表现出较高的细胞毒性对YAC-1和CC531s靶细胞系20中 ，显示出具有更高的数目较小的胞质颗粒20，和表面抗原，诸如LFA-1的12的高表达。另?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
The authors and this work were supported by NIH/NCI grant # R01CA172138 (to SBE).
Materials
| Autoclud Peristaltic pump | |||
| Butterfly needle | OMG | 26G | |
| Butterfly needle | OMG | 21G*3/4" | |
| Syringe | Pic solution | 1 ml | |
| Syringe | Pic solution | 2.5 ml | |
| Syringe | Pic solution | 5 ml | |
| Syringe | Pic solution | 10 ml | |
| Syringe | Pic solution | 25 ml | |
| Blunted-edged forceps | |||
| Scissors | |||
| hemostat | |||
| Tissue forceps | |||
| 22W Fluorescent Daylight Magnifier Lamp |
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