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罗维,艾磊,周越.小鼠巨噬细胞 RAW264. 7 细胞电穿孔转染条件的建立和验证[J].中国实验动物学报,2020,28(2):0.
小鼠巨噬细胞 RAW264. 7 细胞电穿孔转染条件的建立和验证
Establishment and verification of transformation conditions for mouse RAW264. 7 macrophages by electroporation
投稿时间:2019-09-09  
DOI:10. 3969 / j.issn.1005-4847. 2020. 02. 007
中文关键词:  RAW264. 7 细胞  电穿孔  TRIB3 过表达质粒  TRIB3 siRNA
英文关键词:RAW264. 7 cells  electroporation  TRIB3 overexpression plasmid  TRIB3 siRNA
基金项目:
作者单位E-mail
罗维 1.南京体育学院运动健康学院,南京 210014
2. 北京体育大学运动生理学教研室,北京 100084 
luoweihyd@ 163.com 
艾磊 江苏省体育科学研究所,南京 210033  
周越 北京体育大学运动生理学教研室,北京 100084 zhouy@ bsu.edu.cn 
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中文摘要:
      目的 筛选并验证 RAW264. 7 细胞中电穿孔转染 TRIB3 过表达质粒和 TRIB3 siRNA 的条件,建立适用于 RAW264. 7 细胞的最佳电穿孔转染方案。 方法 通过改变脉冲电压、脉冲时长和质粒浓度等条件,将载体为 pCMV6-Entry 的 TRIB3 过表达质粒和 TRIB3 siRNA 转入 RAW264. 7 细胞,24 h 后相差显微镜观察细胞形态, CCK-8法检测细胞存活率,72 h 后 Western Blot 法检测 TRIB3 蛋白表达以验证目的基因表达改变情况。 结果 (1) 以不同脉冲电压转染时,110 和 120 mV 组细胞形态和存活率与对照组无差异,130 和 140 mV 组悬浮细胞较多、细 胞存活率显著低于对照组(P< 0. 01),120、130 和 140 mV 组 TRIB3 蛋白表达显著增加(P< 0. 05);(2)以不同脉冲 时长进行转染时,10 和 20 ms 组细胞形态和存活率与对照组无差异,30 ms 组细胞存活率显著低于对照组(P< 0. 01),20 和 30 ms 组 TRIB3 蛋白表达显著增加(P< 0. 01);(3)以不同质粒浓度进行转染时,2 和 4 μg 组细胞形态 和存活率与对照组无差异,6 μg 组细胞存活率低于对照组(P< 0. 05),4 μg 组 TRIB3 表达显著增加(P< 0. 01);(4) 以 120 mV、20 ms 条件转染不同浓度 TRIB3 siRNA 时,各组细胞形态和存活率均与对照组无差异,10 nmol / L 组 TRIB3 表达仅为对照组的 0. 19 倍( P< 0. 01)。 结论 以 120 mV、20 ms 条件在 RAW264. 7 细胞中电穿孔转染 TRIB3 过表达质粒(每样本 4 μg)和 TRIB3 siRNA(10 nmol / L),在保证较高的细胞存活率的同时能显著改变目的基 因的蛋白表达水平,为后续 RAW264. 7 细胞中质粒转染相关的基础研究提供实验依据和借鉴。
英文摘要:
      Objective To screen and validate the conditions for transforming Tribbles Pseudokinase 3 (TRIB3) overexpression plasmids and siRNA into RAW264. 7 cells by electroporation, to establish the best electroporation scheme for RAW264. 7 cells. Methods By changing the conditions of pulse voltage, pulse duration and plasmid concentration,TRIB3 overexpression plasmid and TRIB3 siRNA were transformed into RAW264. 7 cells. Cell morphology was observed by phase contrast microscopy. Cell viability was detected by CCK-8, and TRIB3 protein expression was detected by Western blot. Results (1) When transformed with different pulse voltages, there was no difference in cell morphology and survival rate between 110 and 120 mV groups and the control group. The cell survival rate in the 130 and 140 mV groups were significantly lower than in the control group (P< 0. 01). TRIB3 protein expressions in the 120, 130 and 140 mV groups were significantly increased than that in the control group (P < 0. 05). ( 2) When transformed with different pulse durations, there was no difference in cell morphology and survival rate between the 10 and 20 ms groups and the control group. The cell survival rate in the 30 ms group was significantly lower than that in the control group (P< 0. 01). The expressions of TRIB3 in the 20 and 30 ms groups were significantly increased than that in the control group (P< 0. 01). (3) When transformed with different plasmid concentrations, the cell morphology and survival rate of the 2 and 4 μg groups were not different from the control group. The cell survival rate of the 6 μg group was lower than that in the control group (P< 0. 05), and the expression of TRIB3 in the 4 μg group was significantly increased than that in the control group (P< 0. 01). (4) When transformed with TRIB3 siRNA at different concentrations, the expression of TRIB3 in the 10 nmol / L group was only 0. 19 times that in the control group ( P < 0. 01). Conclusions In RAW264. 7 cells, the TRIB3 overexpression plasmid ( 4 μg / sample) and TRIB3 siRNA ( 10 nmol / L) were transfected by electroporation under the conditions of 120 mV and 20 ms, which ensured a high cell survival rate and significantly changed the protein expression level of the target gene.
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