人胎儿表皮干细胞的体外分离培养及基因转染  

The in vitro isolation, culture and transfection of human fetal epidermal stem cells

作　　者：丁国斌[1] 陈璧[1] 韩军涛[1] 汤朝武[1] 王波涛[1] 

机构地区：[1]第四军医大学西京医院烧伤科,西安710032

出　　处：《中华烧伤杂志》2003年第1期18-21,共4页Chinese Journal of Burns

摘　　要：目的　探讨人胎儿表皮干细胞体外分离培养的方法以及作为体外基因转染靶细胞的可行性。　方法　利用Ⅳ型胶原快速贴附法分离人胎儿表皮干细胞 ,以人胎儿成纤维细胞条件培养液配制表皮干细胞培养基 ,通过角蛋白 19(K19)和整合素 β1免疫组化染色、细胞周期分析及克隆形成率测定 ,对培养细胞进行鉴定。采用脂质体介导法 ,以含血管内皮细胞生长因子 16 5 (VEFG16 5 )基因片段的真核表达载体 pcDNA3 .1( pcDNA3.1/VEGF16 5 )转染培养细胞 ;采用病毒载体介导法 ,以含报告基因绿色荧光蛋白 (GFP)的重组腺相关病毒载体 (raav/GFP)转染培养细胞。应用免疫组化染色及荧光显微镜观察检测转染效果。　结果　人胎儿表皮干细胞呈明显克隆性生长 ,克隆形成率高 ,G1期细胞比例明显高于普通基底层角质细胞 ,K19和整合素 β1免疫组化染色呈强阳性。pcDNA3 .1/VEGF16 5转染的表皮干细胞VEGF16 5免疫组化染色阳性 ,raav/GFP转染的表皮干细胞呈现强荧光。　结论　利用Ⅳ型胶原快速贴附法及人胎儿成纤维细胞条件培养基 ,可初步实现人胎儿表皮干细胞的分离培养。以脂质体为介导或以腺相关病毒为载体进行人胎儿表皮干细胞的体外基因转染是可行的。Objective To explore the in vitro methods of isolation and culture of human fetal epidermal stem cells (HFESCs) and the feasibility of the cultured cells as the target cells for gene transfection. Methods The HFESCs were isolated by means of type IV collagen rapid adhering method. The culture medium for HFESCs was prepared according to that for human fetal fibroblasts. The cultured cells were identified by immunohistochemistry staining of keratin-19 and integrin-β1, cell cycle analysis and clone forming rate determination. Then the cultured cells were gene transfected in vitro by liposome mediating method in which eukaryon expression vector pcDNA3.1/VEGF165 containing vascular endothelial growth factor 165 (VEGF165) were transfected into cultured cells, or by virus vector mediating method in which recombinant adenovirus accompanied vector (raav) containing green fluorescent protein (GFP) (raav/GFP) were transfected into the cultured cells, respectively. The results of in vitro gene transfection of HFESCs were observed by immunohistochemisty staining and fluorescence microscope. Results HFESCs grew well and formed large clones with higher cloning efficiency and higher ratio of G1 cells than keratinocytes. The cultured cells were strongly positive with immunohistochemistry staining of keratin-19 and integrin-β1. After being gene-transfected by pcDNA3.1/VEGF165, the VEGF165 of HFESCs showed positive immunohistochemistry staining property, while the HFESCs transfected by raav/GFP exhibited strong fluorescence. Conclusion HFESCs could be isolated and cultured in vitro by means of rapid adherence to type IV collagen .It seemed feasible that HFESCs were gene transfected with liposome or adeno-associated virus as the vector. [

关 键 词：人胎儿 表皮干细胞 体外分离 体外培养 基因转染 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]