永生大鼠肝星状细胞系的建立及鉴定

目的建立永生性大鼠肝星状细胞(hepaticstellate cell,HSC)系.方法采用改良胶原酶原位灌注法消化肝脏,经Nycodenz密度梯度离心分离、鉴定成年Sprague-Dawley大鼠的HSC.然后通过细胞克隆建立HSC系(HSC-PQ)并传代培养.结合细胞动力学、光镜、透射电镜及免疫细胞化学技术检测HSC-PQ系的倍增时间、(平滑肌肌动蛋白(α-smoothmuscle actin,α-SMA)及结蛋白表达、细胞外基质(extracellular matrix,ECM)合成等表型特征.结果分离获得的HSC约为2×107细胞/只大鼠,细胞成活率＞95%,纯度＞90%,培养2周后几乎所有HSC均已活化.在此基础上经细胞克隆所得的HSC-PQ系表型类似成纤维细胞,倍增时间约75h,可表达结蛋白、α-SMA以及除Ⅳ型胶原外的多种ECM成分(Ⅰ型胶原、Ⅲ型胶原、纤维连接蛋白、层粘连蛋白等),且传代过程中增殖、ECM表达等特性均保持稳定.该细胞系已在体外培养32代(1a以上),提示其具有永生性.结论已成功建立永生性大鼠HSC系,该细胞系的基本特征与活化的原代HSC相似.

Establishment and identification of a novel immortalized rat hepatic stellate cell line, HSC-PQ

[Objective] To establish and identify a novel immortalized rat hepatic stellate cell (HSC) line.[Methods] Primary HSCs were isolated from a normal adult male Sprague-Dawley rat by in situ perfusion with collagenase Ⅳ and pronase E, and then were purified by single-step density gradient centrifugation with nycodenz.Once they reached total activation in culture, a new immortalized myofibroblast-like HSC line was established through cellular cloning. Its characteristics were identified by means of immunocytochemical staining, light microscopy, transmission electron microscopy, and growth curve analysis. [Results] The novel HSC line, termed HSCPQ, had a doubling time of about 75 hours in the Dulbecco's modified Eagle medium (DMEM) containing 20% fetal bovine serum. Most of the main morphological characteristics of the differentiated primary HSC could be detected in HSC-PQ cell, while functional features of activated HSC such as αsmooth muscle actin, desmin, collagen type I, collagen type Ⅲ, fibronectin, laminin and other extracellular matrix proteins could also be found in it except for collagen type Ⅳ. In contrast, fat droplets and autofluorescence of vitamin A disappeared in the HSC-PQ line. This cell line had been maintained in culture for over 30 passages and more than 1 year with little alternation in biological characteristics. [Conclusions] We had successfully established a new rat HSC line (HSC-PQ). It consistently retained the characteristics of activated primary HSC, and was proved to be immortalized.