Pan Qin, Li Ding-Guo, Lu Han-Ming, Wang Yu-Qing, Zhang Wen-Zhu, Xu Qin-Fang
Department of Gastroenterology, Xinhua Hospital, Shanghai Second Medical University, Shanghai 200092, China.
Hepatobiliary Pancreat Dis Int. 2005 May;4(2):281-4.
Playing a central role in hepatic fibrosis, hepatic stellate cell (HSC) has made itself the major target of research. The limited supply of HSC, however, can not meet the ever growing need of experiment. Establishment and identification of novel immortalized HSC line thus may be urgently required.
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.
The novel HSC line, termed HSC-PQ, 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 alpha-smooth 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.
A new rat HSC line (HSC-PQ) has been successfully established. It consistently retains the characteristics of activated primary HSC, and has proved to be immortalized.
肝星状细胞（HSC）在肝纤维化中起核心作用，已成为主要研究对象。然而，HSC的供应有限，无法满足不断增长的实验需求。因此，迫切需要建立和鉴定新的永生化HSC系。
通过用IV型胶原酶和链霉蛋白酶E原位灌注，从正常成年雄性Sprague-Dawley大鼠中分离原代HSC，然后用Nycodenz进行单步密度梯度离心纯化。当它们在培养中完全活化后，通过细胞克隆建立了一种新的永生化肌成纤维细胞样HSC系。通过免疫细胞化学染色、光学显微镜、透射电子显微镜和生长曲线分析对其特征进行鉴定。
新的HSC系称为HSC-PQ，在含有20%胎牛血清的杜氏改良 Eagle培养基（DMEM）中倍增时间约为75小时。在HSC-PQ细胞中可检测到分化的原代HSC的大多数主要形态特征，除IV型胶原外，活化HSC的功能特征如α-平滑肌肌动蛋白、结蛋白、I型胶原、III型胶原、纤连蛋白、层粘连蛋白和其他细胞外基质蛋白也可在其中发现。相比之下，HSC-PQ系中脂肪滴和维生素A的自发荧光消失。该细胞系已在培养中传代30多次并维持了1年多，生物学特性几乎没有变化。
成功建立了一种新的大鼠HSC系（HSC-PQ）。它始终保留活化原代HSC的特征，并已被证明是永生化的。
