mRNA电转抗CD3抗体刺激的PBMC方法的建立

Establishment of an electroporation method for transfection of mRNA into anti-CD3 antibody stimulated human PBMC

目的建立信使RNA(mRNA)电转抗CD3抗体刺激的外周血单个核细胞(PBMC)的方法,为制备基因修饰CDR3δ移植型γδT淋巴细胞,为肿瘤细胞过继治疗提供新的技术方法。方法以SpeⅠ内切酶消化重组p GEM4Z/EGFP/A64质粒,回收纯化线性化的重组质粒p GEM4Z/EGFP/A64,体外转录成mRNA;在电转参数为500 V500μs、400 V 500μs或300 V 500μs等不同条件下,转染抗CD3抗体刺激的人PBMC;用倒置荧光显微镜观察和流式细胞仪观察和分析绿色荧光蛋白的表达;台盼蓝染色计算不同电转条件下细胞的存活率,以确定mRNA电转最佳条件。结果 p GEM4Z/EGFP/A64质粒经过SpeⅠ内切酶酶切,获得了线性化的质粒;体外转录得到了增强型绿色荧光蛋白(EGFP)mRNA;与其他转染条件相比,在500 V 500μs电转参数下转染EGFP mRNA的PBMC细胞,电转后48 h绿色荧光蛋白表达量较高;与其他转染条件相比,在500 V 500μs电转参数下转染EGFP mRNA的PBMC细胞,电转48 h后绿色荧光蛋白表达的阳性细胞高达77%;台盼蓝染色分析不同电转条件下细胞的存活率,在细胞电转48 h后,500 V 500μs参数电转条件下细胞存活率可以达到85%以上。结论电转参数为500 V 500μs的条件可用于电转mRNA于抗CD3抗体扩增的人PBMC,以制备基因修饰T淋巴细胞。

Objective To establish an electroporation method to transfect mRNA into human PBMCs for the preparation of CDR3-grafted γδ T lymphocytes. Methods Linearized plasmids pGEM4Z/EGFP/A64 containing EGFP cDNA fragment by the digestion with restriction endonuclease Spe I were used as templates for EGFP mRNA transcription. EGFP mRNA was transfected into anti-CD3 antibody-stimulated human PBMC by electroporation instrument under different conditions with 500 V and 500 μs, 400 V and 500 μs or 300 V and 500 μs, respectively. The levels of EGFP expression in the cells electroporated with EGFP mRNA were evaluated through inverted fluorescence microscope and flow cytometry. The survival rates of transfected-cells were measured by trypan blue staining.Results Agarose electrophoresis showed that pGEM4Z/EGFP/A64 plasmids were well linearized by Spe I digestion and EGFP mRNAs were successfully transcribed in vitro. 48 hours after mRNA-transfection, EGFP expression in cells transfected EGFP mRNAs under the condition of 500 V and 500 μs attained 77% which were significantly higher than those in cells transfected EGFP mRNAs under other conditions. The survival rate of transfected cells under the condition of 500 V and 500μs reached up to 85 %. Conclusions The electroporation platform of mRNA transfection into human PBMC with parameters of 500 V and 500 μs is successfully established and may be useful to prepare genetically modified T lymphocytes by mRNA transfection.