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目的 :寻求基因转移中快速检测转染阳性细胞的方法。方法 :采用逆转录病毒介导的基因方法 ,将Ⅰ型单纯疱疹病毒胸苷激酶 (HSV -TK)基因、绿色荧光蛋白 (GFP)基因及抗新霉素 (NeoR)基因插入人外周血单个核细胞 (PBMNC) ,利用GFP在细胞内的表达所产生的绿色荧光 ,经流式细胞仪测定基因转移效率。结果 :被转染细胞以T淋巴细胞为主 ,占 11.39% ,而且被转化的T细胞中CD4 阳性细胞转化率较高 ,占 7.6 1% ,CD8,CD19和CD33 阳性细胞转化率分别为 2 .9% ,2 .1%和 4.72 %。PCR鉴定表明转染的人外周血单个核细胞中含有NeoR基因。结论 :在基因转移中GFP可以为一种标记基因 ,快速检测转染阳性细胞
OBJECTIVE: To find a method for rapid detection of transfected positive cells during gene transfer. Methods: The HSV-TK gene, green fluorescent protein (GFP) gene and NeoR gene were inserted into human peripheral blood mononuclear cells by retrovirus-mediated gene method. Cells (PBMNC), the green fluorescent light generated by the expression of GFP in the cells was used to determine the gene transfer efficiency by flow cytometry. Results: T lymphocytes were predominant in the transfected cells, accounting for 11.39% of all the transfected cells. The percentages of CD4 positive cells in transformed T cells were higher (7.61%), and the rates of CD8, CD19 and CD33 positive cells were 2. 9%, 2.1% and 4.72%. PCR identification showed that the transfected human peripheral blood mononuclear cells contained NeoR gene. CONCLUSION: GFP can be a marker gene in gene transfer and can rapidly detect transfected positive cells