摘要目的 建立肝硬化组织来源纤维母细胞的分离、培养方法,探讨体外培养纤维母细胞的生物学特性.方法 采用组织块贴壁法等细胞培养技术分离培养肝硬化组织来源纤维母细胞,通过显微摄像、细胞计数研究其形态学变化及生长特性,用细胞免疫荧光及Western blotting等方法检测细胞成纤维细胞特异性蛋白1(FSP1)、波形蛋白的表达情况,对所得细胞进行分析鉴定.结果 2h内组织块贴壁良好,1周左右可见组织块周边有单个的胞体较大的肝细胞样细胞游出,2周左右可见梭形或三角形纤维母细胞游出.传代后细胞呈均一的成纤维细胞样,生长潜伏期短,纤维母细胞生长性状稳定,3、4、5代细胞生长曲线基本一致.细胞免疫荧光结果表明,细胞表达FSP1、波形蛋白呈阳性,阳性率分别为(90.6±1.0)％和(91.2±4.1)％.结论 采用组织块贴壁培养法能获得纯度较高的纤维母细胞,此方法简便、高效、稳定,培养的细胞具有良好的细胞生物学特性.

abstractsObjective To establish a method for isolating and culturing fibroblasts from cirrhotic liver in vitro,observe the biological characteristics of fibroblasts.Methods Human fibroblasts from cirrhotic liver were isolated and cultured by adhering to the culture plastic.The morphologic and growth characteristics of the acquired cells were observed by microscopy imaging and cell counting.The expression of FSP-1 and Vimentin of fibroblasts was detected through immunofluorescence and western blotting.Results The tissue blocks were well-adherent to the culture plastic within 2 hours.Hepatocyte-like cells were observed surrounding the blocks within about 1 week,and spindle-shaped fibroblasts were observed within about 2 weeks.After a series of passage,the attached cells became proliferated quickly.The growth curves of the passage 3,4 and 5 were quite similar.The result of immunofluorescence showed the expression of FSP1 and Vimentin was positive.The positive rate was respectively (90.6 ± 1.0) ％ and (91.2-± 4.1) ％.Conclusions Human fibroblasts from cirrhotic liver can be cultured successfully through adherent property.The method for isolation and culture of fibroblasts from cirrhotic liver is convenient,efficient,stable and cultured cells remain naive biological characteristics.