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目的 建立并鉴定肝星状细胞 (HSC)细胞系。 方法 采用肝脏离体胶原酶灌注消化及密度梯度离心的方法分离培养大鼠的HSC ;通过体外长期传代培养后建立 1株细胞系 ,应用细胞形态学观察、细胞倍增时间测定、细胞生长曲线绘制、染色体分析、双层软琼脂培养、免疫细胞化学检测、RT PCR检测、聚硅酮膜培养法观测HSC的收缩等方法对其生物学特性进行研究。 结果 HSC细胞系已传了 78代 ,保持着肌成纤维样细胞的形态学特点。细胞倍增时间为 36 5h ;细胞的染色体众数 4 2条 ,为正常大鼠体细胞的染色体表型 ;双层软琼脂培养未见细胞克隆形成 ;免疫细胞化学检测表明 ,细胞系表达Desmin、GFAP、Ⅰ型胶原、Ⅲ型胶原、α SMA、iNOS、ICAM 1、TGF α ;RT PCR检测表明 ,该细胞系表达TGF β、ICAM 1;聚硅酮膜培养法显示 ,HSC细胞系能在ET 1作用下产生收缩反应。结论 HSC细胞系符合建系标准 ,是 1株新建立的大鼠肝星状细胞系 ,命名为rHSC 99,可作为肝硬化、门静脉高压症实验研究的模型。
Objective To establish and identify a hepatic stellate cell (HSC) cell line. Methods Rat liver HSCs were isolated and cultured by collagenase digestion and density gradient centrifugation in vitro. One cell line was established by long-term subculture in vitro. The cell morphology was observed, the cell doubling time was measured and cell growth curve was drawn. Chromosome analysis, double soft agar culture, immunocytochemical detection, RT PCR detection, polysilicon membrane culture method to observe the contraction of HSC and other methods to study its biological characteristics. Results The HSC cell line has passed 78 generations, maintaining the morphological characteristics of myofibroblast-like cells. The cell doubling time was 36 5 hours. The chromosome number of the cells was 42, which was the chromosome phenotype of somatic cells in normal rats. No colony formation was observed in double soft agar culture. Immunocytochemistry showed that the expression of Desmin and GFAP , TypeⅠcollagen, collagen Ⅲ, α SMA, iNOS, ICAM 1 and TGFα. RT-PCR showed that the cell lines expressed TGFβ and ICAM 1. Silicone membrane culture showed that the HSC cell line could express in ET 1 Under the action of contraction. Conclusion HSC cell line is in line with the establishment of the Department of standards, is a newly established rat hepatic stellate cell line, named rHSC 99, can be used as a model of experimental study of cirrhosis and portal hypertension.