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人外周血单核细胞3种不同转染方法的比较 被引量：6

Comparison of Transfection Efficiency of Monocytes from Human Peripheral Blood by Three Different Methods
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摘要 目的采用3种不同方法转染原代人外周血单核细胞,以寻找一种简便、高效的单核细胞转染方法。方法采用人淋巴细胞分离液从健康人外周血中分离出单个核细胞,锥虫蓝拒染法检测细胞存活率。单个核细胞在孔板内培养2 h后吸弃悬浮的淋巴细胞,获得贴壁的单核细胞。分别采用Lipofectamine 2000介导载体质粒(PEGF-N1),慢病毒(NC-GFP-LV)及腺病毒(Adv-GFP)3种方法转染单核细胞,48 h后在荧光显微镜下观察绿色荧光蛋白(GFP)表达情况,并收集细胞上流式细胞仪检测GFP阳性细胞的比例。结果通过人淋巴细胞分离液分离的单个核细胞的活细胞率>95%,获得的单核细胞数量多,贴壁牢,形态均一。转染48 h后,Lipofectamine 2000介导载体质粒及慢病毒转染的单核细胞无绿色荧光,流式细胞仪检测GFP阳性细胞均<1%,腺病毒转染的单核细胞可见较强绿色荧光,GFP阳性细胞约占10%。结论腺病毒,而不是Lipofectamine 2000或慢病毒,能有效地将外源基因导入单核细胞内表达。 Objective To explore the transfection efficiency of primary monocytes from human peripheral blood by three different methods in order to acquire the method with higher transfection efficiency.Methods Mononuclear cells from human peripheral blood were isolated using human lymphocytes isolation buffer.Cell viability was detected by using Trypan blue staining.Monocytes were obtained by removing the suspending lymphocytes and then transfected by Lipofectamine 2000 with plasmid（PEGF-N1）,adenovirus vector（Adv-GFP）and lentivirus vector（NC-GFP-LV）respectively.After transfection for 48 h,green fluorescence was detected under the fluorescence microscopy and percentage of positive cells was checked by flow cytometry.Results Viability of mononuclear cells isolated by human lymphocytes isolation buffer was over 95%.After removing lymphocytes,there were numerous monocytes which adhered to plate strongly with a uniform shape.No green fluorescence was observed after trasfection of plasmid by Lipofectamine 2000 or lentivirus vector,positive cells were less than 1%,while about 10% monocytes showed strong green fluorescence after transfection with adenovirus vector.Conclusion Adenovirus vector,but not Lipofectamine 2000 or lentivirus,could efficiently transfect exogenous genes into monocytes for expression.
作者 叶双梅 阚淳一 杨丽兰 杨洁 马全富 吴明富 卢运萍 王世宣
机构地区 华中科技大学同济医学院附属同济医院妇产科
出处 《华中科技大学学报（医学版）》 CAS CSCD 北大核心 2011年第2期196-199,共4页 Acta Medicinae Universitatis Scientiae et Technologiae Huazhong
基金 卫生部公益性行业科研专项项目(No.200802159) 教育部高等学校博士点科研基金(No.200804870009) 国家自然科学基金(No.30973148)资助项目
关键词 外周血 单核细胞 腺病毒 慢病毒 基因转染 peripheral blood monocytes adenovirus lentivirus gene transfection
分类号 R349.8 [医药卫生—基础医学]
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参考文献12

  • 1吴鹏,刘映峰,梁东辉,陈允钦.提高人外周血单核细胞分离率的方法探讨[J].实用医学杂志,2008,24(5):707-708. 被引量：16
  • 2Wolfrum N, Mtihlebach M D, Schiule S, et al. Impact of viral accessory proteins of SIVsmmPBj on early steps of infection of quiescent cells[J]. Virology, 2007,364 (2) : 330-341.
  • 3Muhlebach M D,Wolfrum N,Schule S,et al. Stable transduc tion of primary human monocytes by simian lentiviral vector PBj[J]. Mol Ther,2005,12(6) : 1206-1216.
  • 4李涛,满江红,巩伟丽,靳宝锋.人外周血单核细胞的分离和电穿孔法转染[J].生物技术通讯,2010,21(5):673-676. 被引量：5
  • 5Bhattacharjee A,Mishra R S,Feldman G M,et al. In vivo val- idation of signaling pathways regulating human monocyte chemotaxis[J]. J Immunol Methods,2008,330(1/2) :86-95.
  • 6Dong C, Kwas C, Wu L. Transcriptional restriction of human immunodeficiency virus type 1 gene expression in undifferen- tiated primary monocytes[J]. J Virol, 2009, 83 (8):3518-3527.
  • 7Komano Y, Nanki T, Hayashida K, et al. Identification of a human peripheral blood monocyte subset that differentiates into osteoelasts[J]. Arthritis Res Ther,2006,8(5):R152.
  • 8Mountain A. Gene therapy: the first decade[J]. Trends Biotechnol,2000,18(3) :119-128.
  • 9Schneider S D, Rusconi S, Seger R A, et al. Adenovirus-media ted gene transfer into monocyte-derived macrophages of pa- tients with X linked chronic granulomatous disease: ca- vivo correction of deficient respiratory burst[J]. Gene Ther, 1997, 4(6) : 524-532.
  • 10Wirtz S,Becker C,Blumberg R,et al. Treatment of T cell-de- pendent experimental colitis in SCID mice by local administra- tion of an adenovirus expressing IL-18 antisense mRNA[J]. J Immunol,2002,168(1) :411-420.

二级参考文献17

  • 1王福生,金磊,洪卫国,刘明旭,周越塑,张冰,施明,王吉明,雷周云,王哲,冯铁建,侯静,李光汉,曹韵贞.Low Frequencies of CCR5-Δ32 and CCR5-m303,but High Frequencies of CCR2-641 and SDF1-3'A Alleles in Indigenous Ethnic Groups in China's Mainland[J].Chinese Journal of Sexually Transmitted Infections,2002,2(1):7-12. 被引量：5
  • 2Volkman A,Gowans J L.The origin of macrophages from human bone marrow in the rat[J].Br J Exp Pathol,1965,46:62-68.
  • 3van Furth R,Cohn Z A.The origin and kinetics of mononuclear phagocytes[J].J Exp Med,1968,128:415-435.
  • 4van Furth R,Diesselhoff-den Dulk M M C,Mattie H.Quantitative study on the production and kinetics of mononuclear phagocytes during an acute inflammatory reaction[J].J Exp Med,1973,138:1314-1330.
  • 5Serbina N V,Jia T,Hohl T M,et al.Monocyte-mediated defense against microbial pathogens[J].Annu Rev Immunol,2008,26:421-452.
  • 6Martinet W,Schrijvers D M,Kockx M M.Nucleofection as an efficient nonviral transfection method for human mouocytic cells[J].Biotechnol Lett,2003,25(13):1025-1029.
  • 7Gordon S,Taylor P R.Monocyte and macrophage heterogeneity[J].Nat Rev Immunol,2005,5(12):953-964.
  • 8Chen L,Dong W,Zou T,et al.Protein phesphatnse 4 negatively regulates LPS cascade by inhibiting ubiquitination of TRAF6[J].FEBS Lett,2008,582(19):2843-2849.
  • 9Lomagu M A,Yeh W C,Sarosi I,et al.TRAF6 deficiency results in osteopotrosis and defective interleukin-1,CD40,and LPS signaling[J].Genes Dev,1999,13(8):1015-1024.
  • 10Kobayashi T,Walsh M C,Choi Y.The role of TRAF6 in signal transduction and the immune response[J].Microbes Infect,2004,6(14):1333-1338.

共引文献19

同被引文献64

  • 1Dostalova I, Kunesova M, Duskova J, et al. Adipose tissue re- sistin levels in patients with anorexia nervosa [J]. Nutrition, 2006, 22 (10): 977-983.
  • 2He TC, Zhou S, Da Costa LT, etal. A simplified system for generating recombinant adenoviruses [J]. Proc Natl Acad Sci US- A, 1998, 95 (5): 2509-2514.
  • 3Nilsson M, Ljungberg J, Richter J, et ol. Development of an adenoviral vector system with adenovirus serotype 35 tropism; ef- ficient transient gene transfer into primary malignant hematopoi- etic ceils [J]. J Gene Med, 2004, 6(6): 631-641.
  • 4Koizumi N, Mizuguehi H, Hosono T, et al. Efficient gene trans- fer by fiber-mutant adenoviral vectors containing RGD peptide [J]. Biochim Biophys Acta, 2001, 1568(1 ) : 13-20.
  • 5Okada N, Tsukada Y, Nakagawa S, et al. Efficient gene delivery into dendritic cells by fiber-mutant adenovirus vectors [J]. Biochem Biophys Res Commun, 2001, 282( 1 ) : 173- 179.
  • 6Segerman A, Atkinson JP, Marttila M, et al. Adenovirus type 11 uses CD46 as a cellular receptor [J]. J Virol, 2003, 77 (17): 9183-9191.
  • 7Fishelson Z, Donin N, Zell S, et al. Obstacles to cancer im- munotherapy: expression of membrane complement regulatory proteins (mCRPs) in tumors [J]. Mol Immunol, 2003, 40(2-4): 109-123.
  • 8Havenga MJ, Lemckert AA, Ophorst OJ, et al. Exploiting the natural diversity in adenovirus tropism for therapy and prevention of disease [J]. J Virol, 2002, 76(9): 4612-4620.
  • 9Friend DS, Papahadjopoulos D, Debs RJ. Endocytosis and intra- cellular processing accompanying transfection mediated by cation- ic liposomes [J]. Biochim Biophys Acta, 1996, 1278 (1): 41- 50.
  • 10Serbina NV, Jia T, Hohl TM, et ol. Monocyte-mediated defense against microbial pathogens [J]. Annu Rev Immunol, 2008, 26 ( 1 ) : 421-452.

引证文献6

二级引证文献7

华中科技大学学报（医学版）
2011年 第2期

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