重组绿色荧光蛋白腺相关病毒载体转染293细胞和CD34+细胞

南方医科大学学报 ›› 2005, Vol. 25 ›› Issue (01): 26-29.

重组绿色荧光蛋白腺相关病毒载体转染293细胞和CD34⁺细胞

李黎波¹, 冯茹², 周淑芸²

1. 南方医科大学南方医院肿瘤科, 广东广州510515;
2. 南方医科大学血液科, 广东广州510515

出版日期:2005-01-20 发布日期:2005-01-20
基金资助:
收稿日期:2003-4-30。
基金项目:国家自然科学基金(39670826)
作者简介:李黎波(1964- ),男,第一军医大学在读博士研究生,主要从事肿瘤综合治疗研究工作,电话:020-61641657

Transfection of human 293 and CD34⁺ cells with recombinant green fluorescent protein adeno-associated virus

LI Li-bo¹, FENG Ru², ZHOU Shu-yun²

1. 南方医科大学南方医院肿瘤科, 广东广州510515;
2. 南方医科大学血液科, 广东广州510515

Online:2005-01-20 Published:2005-01-20

摘要/Abstract

摘要： 目的研究重组绿色荧光蛋白腺相关病毒载体分别转染293细胞(人胚肾细胞)和人CD34⁺细胞后绿色荧光蛋白的表达情况。方法用CS-3000血细胞分离机分离骨髓单个核细胞,免疫磁性分离仪纯化CD34⁺细胞,重组绿色荧光蛋白腺相关病毒载体分别转染两种细胞,并通过荧光显微镜、流式细胞仪对绿色荧光蛋白的表达进行分析。结果荧光显微镜下两种细胞均可见绿色荧光蛋白表达,流式细胞仪于一定时间内检测293细胞的基因转导效率最高为32.8%,CD34⁺细胞的基因转导效率最高为25%,在所观察的时间内,转染率呈先升高后降低趋势。结论重组绿色荧光蛋白腺相关病毒载体能转染293和CD34⁺细胞,绿色荧光蛋白基因可作为良好的报告基因,为将来的干细胞基因治疗打下良好基础。

Abstract: Objective To investigate the expressions of recombinant green fluorescent protein (GFP) in 293 cells (human embryonic kidney cells) and CD34⁺ cells transfected with adeno-associated virus carrying the gene encoding the recombinant GFP. Method Mononuclear cells from the bone marrow were collected on cell separator CS-3000, and CD34⁺ cells were separated on immunomagnetic MiniMACS columns. CD34⁺ cells and 293 cells were transfected with recombined adeno-associated virus respectively, and the expressions of the GFP were detected by flow cytometry and fluorescence microscopy. Result GFP expression was observed in the two cells under fluorescent microscope. The highest transfection efficiency of the recombined adeno-associated virus was about 32.8% in 293 cells and 25% in CD34⁺ cells, and decreased with the prolongation of transfection time. Conclusion Adeno-associated viral vector encoding recombinant GFP can be transfected into human 293 cells and CD34⁺ cells, which provides a basis for future gene therapy.

中图分类号:

Q786

李黎波¹, 冯茹², 周淑芸². 重组绿色荧光蛋白腺相关病毒载体转染293细胞和CD34⁺细胞[J]. 南方医科大学学报, 2005, 25(01): 26-29.

LI Li-bo¹, FENG Ru², ZHOU Shu-yun². Transfection of human 293 and CD34⁺ cells with recombinant green fluorescent protein adeno-associated virus[J]. Journal of Southern Medical University, 2005, 25(01): 26-29.

参考文献

[1] PE Monahan, RJ Samulski, J Tazelaar, et al. Direct intramuscular injection with recombinant AAV vectors results in sustained expression in a dog model of hemophilia[J]. Gene Ther 1998, 5: 40-9.
[2] Martin C, Yuan T, Ghia Euskirchen, et al. Green fluorescent protein as a marker for gene expression[J]. Science, 1994, 263(11): 802-5.
[3] Douglas CP, Virginia K, Wiliam WW, et al. Primary structure of the Aecquorea victoria green fluorescent protein [J]. Gene, 1992, 111(2):229-33.
[4] 胡贵方,吴小兵,俞守义,等.含乙型肝炎表面抗原基因重组腺相关病毒的构建及其基因的表达和功能初步研究[J].第一军医大学学报,2003,23(6):553-6.Hu GF, Wu XB, Yu SY, et al. Construction of recombinant adeno-associated virus carrying hepatitis B surface antigen gene and preliminary study of the gene expression and function [J]. J First Mil Med Univ/Di Yi Jun Yi Da Xue Xue Bao, 2003, 23(6): 553-6.
[5] Bartlett JS, Jude SR, Mccown TJ. Selective and rapid uptake of adeno-associated virus type 2 in brain[J]. Human Gene Ther, 1998, 9:1181-6.
[6] 陈捷,周淑芸,冯茹,等.抑制性消减杂交技术检测骨髓CD34⁺细胞基因差异表达[J].第一军医大学学报,2002,22(9):823-5.Chen J, Zhou SY, Feng R. Identification of differentially expressed genes in bone marrow CD34⁺ cells by suppression subtractive hybridization[J]. J First Mil Med Univ/Di Yi Jun Yi Da Xue Xue Bao,2002, 22(9): 823-5.
[7] 胡舜英,冯茹,李黎波,等.腺相关病毒与人多药耐药基因重组载体的构建及其在NIH3T3细胞中的表达[J].中国生物化学与分子生物学报,2000,16(3):326-9.Hu SY, Feng R, Li LB, et al. Construction of recombinant AAVMDR1 and expression in NIH3T3 cells[J]. Chin J Biochem Mol Biol, 2000, 16(3):326-9.
[8] Nathwani AC, Hanawa H, VandergriffJ, et al. Efficient gene transfer into human cord blood CD34⁺ cells and the CD34⁺CD38-subset using highly purified recombinant adeno-associated vira vector preparations that are free of helper virus and wild-type AAV [J]. Gene Ther, 2000, 7(3): 183-95.

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