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目的优化聚乙烯亚胺(polyethylenimine,PEI)介导的细胞转染以提高目的基因在细胞中的表达强度。方法根据PEI/DNA不同的质量比(0:1、0.5:1、1:1、2:1、3:1、4:1、5:1、6:1、7:1、8:1、9:1、10:1)利用凝胶阻滞分析结合的情况;不同质量比PEI/DNA形成的复合物对NIH-3T3细胞的转染通过细胞表达强度获得最佳PEI/DNA质量比;比较实验组1(PEI/DNA质量比2:1)、实验组2(重复PEI/DNA质量比2:1)及实验组3(PEI/DNA质量比4:2)3种转染方案,探讨重复转染的方法是否提高细胞表达强度。结果①通过凝胶阻滞分析PEI/DNA能够稳定结合的质量比是1:1～10:1;②PEI/DNA复合物对NIH-3T3细胞进行转染,其荧光表达强度当PEI/DNA(质量比)≤2时随着PEI的增加而提高,当PEI/DNA(质量比)>2时,荧光表达强度反而降低;③采用重复转染的方法实验组2细胞荧光表达强度(24.08±0.28)%明显高于实验组1(8.97±4.02)%和实验组3(14.24±2.68)%(P<0.05)。结论在PEI/DNA(质量比)=2的条件下,PEI/DNA复合物转化NIH-3T3细胞的效果比较理想,重复转染可以提高细胞转染后荧光表达强度。
Objective To optimize the cell transfection mediated by polyethylenimine (PEI) in order to improve the expression of target gene in cells. Methods According to different PEI / DNA mass ratios (0: 1, 0.5: 1, 1: 1, 2: 1, 9: 1, 10: 1). The best mass ratio of PEI / DNA was obtained by transfecting NIH-3T3 cells with different mass ratios of PEI / DNA complexes. Three transfection protocols of experimental group 1 (PEI / DNA mass ratio 2: 1), experimental group 2 (PEI / DNA mass ratio 2: 1) and experimental group 3 (PEI / DNA mass ratio 4: 2) The method of transfection increases the intensity of cell expression. Results ① The mass ratio of PEI / DNA to stably bind was 1: 1 to 10: 1 by gel block analysis; ② NIH-3T3 cells were transfected with PEI / DNA complex, and the fluorescence intensity of PEI / (P <0.05), the fluorescence intensity of the 2 cells increased with the increase of PEI when PEI / DNA (mass ratio) was> 2; (2) The fluorescence intensity of experimental group was 24.08 ± 0.28 % Was significantly higher than that of experimental group 1 (8.97 ± 4.02)% and experimental group 3 (14.24 ± 2.68)% (P <0.05). CONCLUSION: PEI / DNA complex can be transformed into NIH-3T3 cells under the condition of PEI / DNA (mass ratio) = 2, and the transfection can increase the fluorescence intensity of transfected NIH-3T3 cells.