质粒DNA转染人PBMC电穿孔条件的影响因素
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摘要
目的分析影响质粒转染人PBMC电穿孔效率的不同因素。方法从人外周血中分离得到PBMC,利用携带荧光标记的质粒进行电穿孔转染。分别比较不同的电压、脉冲时间、电转缓冲液、细胞培养状态、质粒质量对转染效率和细胞活率的影响。结果在一定范围内适当增高电压会增加细胞转染效率,但会降低细胞活率;脉冲时间达到一定数值后,转染效率才会有明显的升高;电转缓冲液对于电转后细胞存活率至关重要;未活化的PBMC转染效率低于经刺激活化的PBMC,但细胞活率却高于后者;质粒纯度和质量显著影响电穿孔转染效果,利用全波长光谱扫描仪可对质粒的杂质进行有效的检测,质粒中内毒素的含量显著影响PBMC电转后的存活率。结论对质粒转染人PBMC的电穿孔条件进行了详细的分析与探索,阐明了影响原代淋巴细胞转染的多种因素,对于有效提高人原代淋巴细胞转染效率具有重要意义,为基于淋巴细胞的肿瘤免疫研究提供有益参考。
Objective To analyze the influencing factors of electroporation conditions of human peripheral blood mononuclear cells(PBMCs) transfected with plasmid DNA.Methods Human PBMCs were isolated from peripheral blood and transfected with plasmids expressing fluorescent protein. The voltage,pulse time,electroporation buffer,cell culture status,and plasmid quality were evaluated based on transfection efficiency and cell viability. Results The transfection efficiency was increased with the increase of voltage in a certain range,but simultaneously the cell survival rate was decreased. When the pulse time reached a certain value,the transfection efficiency was significantly increased. Electrotransfer buffer was critical for cell viability after electroporation. The transfection efficiency in inactivated PBMCs was lower than that of activated PBMCs,but the cell viability was higher than the latter. The purity and quality of the plasmid were crucial for the transfection of PBMCs. The impurities in plasmids were effectively estimated by full-wavelength spectral scanner. In addition,endotoxin content in plasmids significantly affected the survival rate of PBMCs after electroporation. Conclusion This study analyzes the influencing factors of electroporation conditions of human PBMCs transfected with plasmid DNA in detail,which may provide references for overcoming the problem of human primary lymphocyte transfection and the tumor immunology research based on lymphocytes.
Objective To analyze the influencing factors of electroporation conditions of human peripheral blood mononuclear cells(PBMCs) transfected with plasmid DNA.Methods Human PBMCs were isolated from peripheral blood and transfected with plasmids expressing fluorescent protein. The voltage,pulse time,electroporation buffer,cell culture status,and plasmid quality were evaluated based on transfection efficiency and cell viability. Results The transfection efficiency was increased with the increase of voltage in a certain range,but simultaneously the cell survival rate was decreased. When the pulse time reached a certain value,the transfection efficiency was significantly increased. Electrotransfer buffer was critical for cell viability after electroporation. The transfection efficiency in inactivated PBMCs was lower than that of activated PBMCs,but the cell viability was higher than the latter. The purity and quality of the plasmid were crucial for the transfection of PBMCs. The impurities in plasmids were effectively estimated by full-wavelength spectral scanner. In addition,endotoxin content in plasmids significantly affected the survival rate of PBMCs after electroporation. Conclusion This study analyzes the influencing factors of electroporation conditions of human PBMCs transfected with plasmid DNA in detail,which may provide references for overcoming the problem of human primary lymphocyte transfection and the tumor immunology research based on lymphocytes.
引文
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[16] KIM J A,Cho K,Shin M S,et al.A novel electroporation method using a capillary and wire-type electrode[J].Biosens.Bioelectron,2008,23(9):1353-1360.
[17] CHANG Lingqian,LI Lei,SHI Junfeng,et al.Micro-/nanoscale electroporation[J].Lab Chip,2016,16(21):4047-4062.
[18] PAGANIN-GIOANNI A,BELLARD E,ESCOFFRE J M,et al.Direct visualization at the single-cell level of si RNA electrotransfer into cancer cells[J].Proc Natl Acad Sci USA,2011,108(26):10443–10447.
[19] RIBEIRO S,MAIRHOFER J,MADEIRA C,et al.Plasmid DNA size does affect nonviral gene delivery efciency in stem cells[J].Cell Reprogram,2012,14(2):130-137.
[20] HENDEL A,BAK R O,CLARK J T,et al.Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells[J].Nat Biotechnol,2015,33(9):985-989.
[2] GODBEY W T,WU K K,MIKOS A G.Poly(ethylenimine)-mediated gene delivery affects endothelial cell function and viability[J].Biomaterials,2001,22(5):471-480.
[3] STRAUSS B E,COSTANZI-STRAUSS E.Combating oncogene activation associated with retrovirus-mediated gene therapy of x-linked severe combined immunodeficiency[J].Braz J Med Biol Res,2007,40(5):601-613.
[4] SARAF A,MIKOS A G.Gene delivery strategies for cartilage tissue engineering[J].Adv Drug Deliv Rev,2006,58(4):592-603.
[5] NEELAPU S S,LOCKE F L,BARTLETT N L,et al.axicabtagene ciloleucel CAR T-cell therapy in refractory large B-cell lymphoma[J].N Engl J Med,2017,377(26):2531-2544.
[6] SEGALINY A I,LI Guideng,KONG Lingshun,et al.Functional TCR T cell screening using single-cell droplet microfluidics[J].Lab Chip,2018,18(24):3733-3749.
[7] JUNE C H,O'CONNOR R S,KAWALEKAR O U,et al.CAR T cell immunotherapy for human cancer[J].Science,2018,359(6382):1361-1365
[8] KIM M Y,YU K R,KENDERIAN S S,et al.Genetic inactivation of CD33 in hematopoietic stem cells to enable CAR T cell immunotherapy for acute myeloid leukemia[J].Cell,2018,173(6):1439-1453.
[9] LESUEUR L L,MIR L M,ANDRE F M.Overcoming the specific toxicity of large plasmids electrotransfer in primary cells in vitro[J] .Mol Ther Nucleic Acids,2016,8(5):e291-e301.
[10] 张放,周载鑫,张丹丹,等.电穿孔法转染人原代成纤维细胞条件的优化[J].第二军医大学学报,2017,38(10):1311-1314.
[11] 张高丽,隋娟,刘喜红,等.Jurkat T 细胞电穿孔转染条件的优化[J].新乡医学院学报,2015 ,32 ( 8 ) :725-727.
[12] 李涛,满江红,巩伟丽,等.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.
[13] ROTH T L,PUIG-SAUS C,YU R,et al.Reprogramming human T cell function and specificity with non-viral genome targeting [J].Nature,2018,559(7714):405-409.
[14] ARGUS F,BOYD B,BECKER S M.Electroporation of tissue and cells:a three-equation model of drug delivery[J].Comput Biol Med,2017 ,84:226-234.
[15] BUIJS M,ZONDERVAN P J,DE BRUIN D M.Feasibility and safety of irreversible electroporation (IRE) in patients with small renal masses:results of a prospective study[J].Springer Berlin Urol Oncol,2019,37(3):183.e1-183.e8.
[16] KIM J A,Cho K,Shin M S,et al.A novel electroporation method using a capillary and wire-type electrode[J].Biosens.Bioelectron,2008,23(9):1353-1360.
[17] CHANG Lingqian,LI Lei,SHI Junfeng,et al.Micro-/nanoscale electroporation[J].Lab Chip,2016,16(21):4047-4062.
[18] PAGANIN-GIOANNI A,BELLARD E,ESCOFFRE J M,et al.Direct visualization at the single-cell level of si RNA electrotransfer into cancer cells[J].Proc Natl Acad Sci USA,2011,108(26):10443–10447.
[19] RIBEIRO S,MAIRHOFER J,MADEIRA C,et al.Plasmid DNA size does affect nonviral gene delivery efciency in stem cells[J].Cell Reprogram,2012,14(2):130-137.
[20] HENDEL A,BAK R O,CLARK J T,et al.Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells[J].Nat Biotechnol,2015,33(9):985-989.
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