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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 investigate the method of human fetal epidermal stem cells isolated and cultured in vitro and the feasibility of transfecting target cells in vitro. Methods Human fetal epidermal stem cells were isolated by rapid attachment of type Ⅳ collagen and cultured in conditioned medium of human fetal fibroblasts. The expression of keratin 19 (K19) and integrin β1 were detected by immunohistochemistry and cell cycle analysis Clone formation rate was measured and cultured cells were identified. The recombinant plasmid pcDNA3.1 (pcDNA3.1 / VEGF165) was transfected into the cultured cells by liposome-mediated method using the vector containing VEFG16 5 gene. The cells were transfected with recombinant adeno-associated virus vector (raav / GFP) containing reporter green fluorescent protein (GFP). Immunohistochemical staining and fluorescence microscopy were used to detect the transfection efficiency. Results Human fetal epidermal stem cells showed obvious clonal growth, clonal formation rate was high, the proportion of cells in G1 phase was significantly higher than that of common basal layer keratinocytes, K19 and integrin β1 immunohistochemical staining was strongly positive. pcDNA3.1 / VEGF165 transfected epidermal stem cells VEGF16 5 immunohistochemical staining, raav / GFP transfected epidermal stem cells showed strong fluorescence. Conclusion The rapid adherence of type Ⅳ collagen and human fetal fibroblasts conditioned medium can initially achieve the isolation and culture of human fetal epidermal stem cells. In vitro gene transfection of human fetal epidermal stem cells with liposome-mediated or adeno-associated virus as a carrier is feasible.