皮肤成纤维细胞体外长期培养及肝向分化研究  

Establishment of the limited cell line of skin fibroblast and their differentiation to hepatic cells

作　　者：朱争艳[1] 杜智[1] 骆莹[1] 黎娇[1] 孙琳琳[1] 王鹏[1] 刘彤[1] 高英堂[1] 

机构地区：[1]天津市第三中心医院；天津市人工细胞重点实验室；天津市再生医学与疾病生物治疗工程研究中心实验室；天津市再生医学与疾病生物治疗工程研究中心,300170

出　　处：《国际生物医学工程杂志》2011年第5期284-287,292,I0004,共6页International Journal of Biomedical Engineering

基　　金：天津市发改委资助项目【津发改高技（2006）26号】

摘　　要：目的本研究通过体外长期培养皮肤成纤维细胞，试图建立此种细胞的长期培养体系，探讨其向肝细胞分化的潜能。方法从人胎上臂取皮肤组织，分离培养成纤维细胞。采用免疫细胞化学法及流式细胞术检测细胞的CD34、CD90、CD105等细胞表型；染色体分析及软琼脂克隆形成实验鉴定细胞的生物学特性。利用肝细胞生长因子（HGF）、成纤维细胞生长因子4（FGF4）、抑瘤素（OSM）等细胞因子对笫3～30代细胞进行肝细胞诱导分化，采用免疫细胞化学方法对诱导和未诱导的细胞进行白蛋白ALB、细胞角蛋白CK18、CK19等免疫学检测，PAS法糖原染色，RT—PCR法检测甲胎蛋白（AFP）和白蛋白（ALB）mRNA表达。结果皮肤成纤维细胞表达CD29、CD49f、HLA—I、CD105等间充质细胞标志，几乎不表达CD90。在肝细胞诱导条件培养基中培养，成纤维细胞转化为类肝细胞的比例约为5％。此种细胞可任体外长期稳定培养，传50余代，保持正常核型，无致瘤性。结论皮肤成纤维细胞具有间充质干细胞的特性，体外能够诱导分化为类肝细胞。Objective To establish the long-term cuhure system for fetal skin fibroblast by performing long time in vitro cultivation of the cells, and study the potential of its differentiation to hepatocytes. Methods Fibroblast was isolated from human fetus skin tissue. Surface phenotypes of cells were detected by ICC and FCM, and biological characteristics were analyzed by the karyotype analysis and soft agar colony formation observation. ALB ,CK18 ,CKI9 were detected by ICC, glycogen stain by PAS, AFP and ALB mRNA by RT-PCR after P3-30 cells were induced differentiation by cytokines of HGF, FGF4 and OSM. Results CD29, CD49f, HLA-I and CD105 were highly expressed while CD90 hardly in skin fibroblast. The rate of induced differentiation of fibroblast into hepatocyte-like cells was approximately 5%. The cells could be cultured in vitro for almost 50 passages with normal kalyotype and no oncogenic and immortalized characteristics. Conclusion The skin fibroblast possesses the characteristic of mesenchymal stem cell and can be induced into hepatocyte-like cell in vitro.

关 键 词：成纤维细胞 诱导分化 类肝细胞 

分 类 号：R[医药卫生]