ISOLATED HEPATIC LIPOCYTES AND KUPFFER CELLS FROM NORMAL HUMAN LIVER - MORPHOLOGICAL AND FUNCTIONAL-CHARACTERISTICS IN PRIMARY CULTURE
ISOLATED HEPATIC LIPOCYTES AND KUPFFER CELLS FROM NORMAL HUMAN LIVER - MORPHOLOGICAL AND FUNCTIONAL-CHARACTERISTICS IN PRIMARY CULTURE
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DOI：
10.1002/hep.1840150211
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发表时间：
1992-02-01
期刊：
影响因子：
13.5
通讯作者：
YAMASAKI, G
中科院分区：
文献类型：
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作者：
FRIEDMAN, SL;ROCKEY, DC;YAMASAKI, G
从啮齿类动物中分离肝脂肪细胞（Ito、星状或脂肪储存细胞）技术的发展有助于确定其在肝脏维生素 A 储存和纤维形成中的作用。在这项研究中，我们开发了一种从正常人肝脏楔形切片中纯化脂肪细胞和库普弗细胞的方法，并检查了它们在原代培养中的特性。收获但未用于移植的供体肝脏切片（400至600克）用威斯康星大学溶液原位灌注，并在48小时内用于脂肪细胞分离。通过用 L-15 盐、链霉蛋白酶和胶原酶通过几个大血管对楔形物进行导管灌注，然后进行 Larex 密度梯度离心来分离细胞。将脂肪细胞铺在未包被的塑料或基底膜样凝胶上。每克肝脏的脂肪细胞和库普弗细胞产量分别为 2.3 +/- 0.6 x 10(5) 和 8.6 +/- 1.4 x 10(5) 个细胞 (n = 5)。根据维生素 A 自发荧光评估，脂肪细胞纯度为 91%；根据荧光葡萄球菌摄取测定，库普弗细胞纯度为 83%。在塑料上培养的脂肪细胞在 48 至 72 小时内扩散，显示出比同类大鼠细胞稍微更异质的类视黄醇液滴大小；在基底膜凝胶上，细胞保持聚集并呈球形，偶尔有纺锤状延伸。通过免疫细胞化学分析，塑料上的脂肪细胞表达 I 型和 III 型前胶原、IV 型胶原和层粘连蛋白，通过 RNAse 保护、Northern 印迹和聚合酶链式反应分别表达 I、III 和 IV 型前胶原信使 RNA。 7 天时脂肪细胞的透射电子显微镜显示出明显的粗面内质网和收缩丝。扫描电子显微镜显示细胞表面光滑，细胞膜下方有核周液滴。随着在塑料上继续原代培养（超过 7 天），细胞出现“激活”（即扩散增加和类视黄醇液滴减少），并根据核放射自显影和 [H-3] 胸苷掺入评估开始增殖。通过扫描电子显微镜观察早期原代培养物中的库普弗细胞显示出明显的膜皱褶和片状伪足。总之，我们建立了一种可重复的人脂肪细胞和库普弗细胞的分离和原代培养方法。
The development of techniques for isolating hepatic lipocytes (Ito, stellate or fat-storing cells) from rodents has been instrumental in defining their role in hepatic vitamin A storage and fibrogenesis. In this study, we developed a method for the purification of lipocytes and Kupffer cell from wedge sections of normal human liver and examined their properties in primary culture. Sections of donor liver (400 to 600 gm) harvested but not used for transplantation were perfused in situ with University of Wisconsin solution and used for lipocyte isolation within 48 hr. Cells were isolated by catheter perfusion of the wedge through several large vessels with L-15 salts, Pronase and collagenase, followed by Larex density gradient centrifugation. Lipocytes were plated on either uncoated plastic or a basement membrane-like gel. Lipocyte and Kupffer cell yields were 2.3 +/- 0.6 x 10(5) and 8.6 +/- 1.4 x 10(5) cells, respectively, per gram of liver (n = 5). Lipocyte purity was 91% as assessed by vitamin A autofluorescence, and Kupffer cell purity was 83% as determined by uptake of fluorescinated staphylococci. Lipocytes cultured on the plastic spread within 48 to 72 hr, displaying slightly more heterogeneous retinoid droplet size than comparable rat cells; on a basement-membrane gel, the cells remained aggregated and spherical with occasional spindlelike extensions. Lipocytes on plastic expressed procollagens I and III, collagen IV and laminin by immunocytochemistry, and types I, III and IV procollagen messenger RNAs by RNAse protection, Northern blot and polymerase chain reaction, respectively. Transmission electron microscopy of lipocytes at 7 days demonstrated a prominent rough endoplasmic reticulum and contractile filaments. Scanning electron microscopy revealed a smooth cell surface with perinuclear droplets beneath the cell membrane. With continued primary culture on plastic (more than 7 days), cells appeared "activated" (i.e., increased spreading and diminished retinoid droplets) and began proliferating as assessed by nuclear autoradiography and [H-3]thymidine incorporation. Kupffer cells observed by scanning electron microscopy in early primary culture displayed prominent membrane ruffling and lamellipodia. In summary, we have established a reproducible method for the isolation and primary culture of human lipocytes and Kupffer cells.