慢病毒载体介导成纤维细胞高效稳定表达绿色荧光蛋白

试验尝试建立稳定表达外源基因的人成纤维细胞系。取成年男性包皮皮肤的皮下组织，分离培养人皮肤成纤维细胞，分别采用脂质体和慢病毒载体介导转染人皮肤成纤维细胞表达绿色荧光蛋白。结果显示，来自成年人包皮皮下组织的成纤维细胞呈梭形，具有快速的增殖和稳定的生长性能。脂质体介导转染的人皮肤成纤维细胞绿色荧光蛋白表达不稳定，阳性细胞表达率低，转染后的人成纤维细胞变得更为细长，细胞生长速度降低。慢病毒载体介导人皮肤成纤维细胞高效稳定表达绿色荧光蛋白，转染后细胞生长性能和形态没有变化，经过多次传代和冻存复苏对人皮肤成纤维细胞绿色荧光蛋白的表达没有影响，通过流式细胞仪对慢病毒介导表达绿色荧光蛋白的人皮肤成纤维细胞检测显示，绿色荧光蛋白表达阳性率为99.85%，并且表达绿色荧光蛋白的人皮肤成纤维细胞细胞均一程度为76.05%。试验证实了慢病毒载体能够高效稳定介导人皮肤成纤维细胞表达绿色荧光蛋白。

Expression of EGFP Gene in Fibroblasts Mediated by Lentiviral Vector

In order to study dedifferentiation and cell orientation in the body of human adult cell, adult human fibroblast lines that stably expressed exterior gene were established. Adult male foreskin subcutaneous tissues apart from epidermis and hypodermis were taken by surgery operation and human foreskin fibroblasts were isolated and cultured, then enhanced green fluorescence protein (EGFP) in human fibroblasts were expressed by lipofectamine and lentiviral vectors, respectively. Results showed that human fibroblasts had shuttle-like image and stable growth characteristics. EGFP was unsteadily expressed in human fibroblasts, positive cells in total fibroblasts were very low, human fibroblasts became thinner and had slower growth characteristic after transfected by lipofectamine. In contrast, EGFP was steadily expressed in human fibroblasts, positive cells in total fibroblasts was very high, and fibroblasts had rapid proliferation and stable growth characteristics that were not affected by continous cell passage and cell frozen-thawn after transfected by lentiviral vectors. In addition, EGFP positive fibroblasts in total cells were 99.85% and homogeneous fibroblasts in total cells were 76.05% by flowcytometer assay. It is showed that expression of EGFP gene in human fibroblasts mediated by lentiviral vector was high efficient and stable.