永生大鼠肝星状细胞系的建立及鉴定  

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

作　　者：潘勤[1] 李定国[1] 汪余勤[1] 徐芹芳[1] 

机构地区：[1]上海第二医科大学附属新华医院消化内科,上海市200092

出　　处：《世界华人消化杂志》2004年第6期1337-1340,共4页World Chinese Journal of Digestology

基　　金：上海市科委基金资助项目;No.004119047~~

摘　　要：目的:建立永生性大鼠肝星状细胞(hepatic stellate 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、α平滑肌肌动蛋白(α-smooth muscle 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 wk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),且传代过程中增生、ECM表达等特性均保持稳定.该细胞系已在体外培养32代(1 a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.AIM: To establish and identify a novel immortalized rat hepatic stellate cell (HSC) line. METHODS: Primary HSCs were isolated from the liver of adult male Sprague-Dawley rats by a combination of pronase-collagenase perfusion and density gradient centrifugation. Then a new HSC line, being HSC-PQ, was established, cultured, and passaged by way of cellular clone. Furthermore, cellular dynamics, light microscopy, transmission electron microscopy, and immunocytochemistry were employed to investigate characteristics of the HSC line. RESULTS: About 2×107 HSCs could be harvested from a Sprague-Dawley rat with the live rate over 95% and purity over 90%. Afterwards, HSC-PQ line was obtained on the basis of total activation of primary HSCs. The phenotype of HSC-PQ cells resembled that of fibroblasts. Firstly, the existence of a-SMA as well as desmin in these cells exhibited their HSC-derived-myofibroblast identity clearly. Secondly, both the doubling time of about 75 hours, and the stable expression of extracellular matrixs including collagen type Ⅰ, collagen type Ⅲ, fibronectin, laminin, etc. showed the fibroblast-like-characteristics of HSC-PQ line. But collagen Ⅳ could not be detected in cytoplasm. In addition, maintaining over one year, 32 passages of the cell line might demonstrate its immortalisation. CONCLUSION: We have established a new immortalized rat HSC line (HSC-PQ), which shares most of the characteristics with primary activated rat HSCs.

关 键 词：大鼠 肝星状细胞系 HSC 细胞动力学 免疫细胞 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]