摘要
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 new rat hepatic stellate cell line (rHSC\|99). Methods Liver in vivo perfusion of collagenase and density gradient centrifugation were used to isolate hepatic stellate cell from a normal rat.Cells were maintained in culture for multiple passages.Cell proliferation and morphological characterization were studied on 60 passage of HSC.Immunocytochemistry and RT\|PCR were used to detect the expression of demin,GFAP,α\|SMA,collagen type Ⅰ and Ⅲ,iNOS,ICAM\|1,TGF\|α,IL\|10 and TGF\|α in 60 passage of HSC.Sixty passage cells were cultured on silicone membrane to investigate the cell contraction.Gimsa stain method was used to analyze karyotype. Results rHSC\|99 cell has undergone 78 passages.It's morphological characteristics resemble myofibroblast cell.The cell doubing time is 36\^5 hours.Immunocytochemistry showed that Demin,GFAP,α\|SMA,collagen type Ⅰ and Ⅲ,iNOS,ICAM\|1,TGF\|α were expressed in rHSC\|99 cells.RT\|PCR analysis showed that TGF\|β and ICAM\|1 were expressed in rHSC\|99 cells.Cell contraction was induced by ET\|1.Cell clone was not formed after 14 days culture in soft agar.rHSC\|99 cells displayed normal rat karyotype with 42 chromosomes in modal number.Conclusion\ rHSC\|99 cell line exhibits characteristics of activated hepatic stellate cell.It will be a useful tool to study the pathogenesis of liver cirrhosis and portal hypertension.\;[
出处
《解剖学报》
CAS
CSCD
北大核心
2003年第3期269-274,共6页
Acta Anatomica Sinica
基金
国家自然科学基金资助项目 ( 3 9970 72 2 )
