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rAAV2载体介导GFP基因转染树突状细胞

GFP gene transfection of dendritic cells mediated by recombinated adeno-associated virus
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摘要 目的:在诱导人骨髓CD34+细胞分化成树突状细胞(dendritic cells,DCs)的基础上,探讨2型重组腺相关病毒(Type2recombinant adeno-associated virus,rAAV2)载体介导绿色荧光蛋白(green fluo-rescent protein,GFP)基因转染DCs的条件。方法:采用免疫磁珠法分离纯化人骨髓来源的CD34+细胞。在含有IL-4和GM-CSF的培养体系中体外诱导CD34+细胞生成DCs,于第5天加入TNF-α诱导DCs成熟,在不同时间用rAAV2载体介导GFP基因转染未成熟和成熟的DCs。通过电子显微镜和流式细胞仪鉴定树突状细胞,荧光显微镜及流式细胞仪检测GFP的表达。结果:人骨髓CD34+细胞经诱导分化后,在光镜及透射电镜下均可观察到DCs的形态特征,流式细胞仪检测到DCs表型;荧光显微镜及流式细胞仪检测细胞培养第3天、第5天和加入TNF-α后的rAAV2-GFP转染率分别为0.45%,13.54%和0.25%。结论:本实验中所采用的培养体系可成功将人骨髓CD34+细胞诱导分化成DCs,rAAV2/GFP转染DCs效率随细胞诱导分化时间的延长而增高,且转染未成熟DCs的效率高于转染成熟DCs。 Objective To investigate the transfection condition of Type 2 recombinant adeno-associated virus （ rAAV2 ） in human dendritic cells （DCs） which were induced from the bone marrow CD34^＋ hematopoietic stem/progenitor cells. Methods CD34^＋ hematopoietic stem/progenitor cells were purified from the bone marrow mononuclear cells by immunomagnetic beads, and the cells were cultured with IL-4 and GM-CSF and maturated by TNF-α on the 5th day. The rAAV2 /GFP was transfected into the induced cells at different time. The DCs were identified by electronic microscope. The expression of GFP was evaluated by flow cytometry and fluorescence microscope. Results The DCs were induced successfully. The typical morphologic characteristics of DCs were observed under the light microscope and transmission electronic microscope, and the typical phenotypes of DCs could be detected by flow cytometry. The expression rate of GFP gene on the 3 rd day, the 5 th day and after adding TNF-α was 0.45% , 13.54% , and 0. 25% , respectively. Conclusion DCs can be induced from the human bone marrow CD34^＋ hematopoietic stem/progenitor cells, and infected with the rAAV2 /GFP successfully. The longer the induction time of DCs, the higher the trans-fection efficiency of DC. The transfection efficiency of immature DC is higher than that of mature DC.
作者 周小莹 暨明 刘君彦 谭孟群
机构地区 中南大学基础医学院生理学系血液研究室 上海复旦张江生物公司
出处 《中南大学学报（医学版）》 CAS CSCD 北大核心 2007年第5期742-746,共5页 Journal of Central South University ：Medical Science
基金 国家863计划项目(2005AA2Z3G10) 高校博士点基金资助项目(20040533031) 湖南省卫生厅科研基金(C2006-001)~~
关键词 重组腺相关病毒2型 GFP 树突状细胞 基因转导 adeno-associated virus green fluorescent protein dendritic cells gene transfection
分类号 Q78 [生物学—分子生物学]
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参考文献14

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中南大学学报（医学版）
2007年 第5期

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