摘要
目的观察NucleofectorTM电转染技术对人胚肺二倍体成纤维细胞(MRC-5)衰老进程的影响。方法取年轻代数的MRC-5细胞(30代),分为3组,第1组为正常培养对照组,第2组为未加质粒电转对照组,第3组为空质粒EGFP-N1电转组。采用NucleofectorTM电转仪配合试剂盒用U-023程序进行电转,计算细胞衰老相关SA-β-半乳糖苷酶(gal)阳性率,分别采用逆转录-聚合酶链反应(RT-PCR)和Western印迹法分别测定p16mRNA、P16蛋白的表达。结果①第1组的SA-β-gal阳性率为(9.01±0.65)%,显著低于第2组的(32.00±0.31)%和第3组的(31.13±1.62)%(P值均<0.01),后两组间的差异无统计学意义(P>0.05)。②第2组、第3组的p16mRNA、P16蛋白均显著高于第1组(P值均<0.05),第2组与第3组间的差异无统计学意义(P值均>0.05)。结论NucleofectorTM电转方法可促进人MRC-5的衰老,此作用可能是通过上调P16蛋白的表达来实现的。
Objective To investigate the influence NucleofectorTM electrotransfection on senescence of human embryo lung fibroblasts （MRC-5 cells）. Methods MRC-5 cells of the 30th passage were divided into 3 groups： normal group （group 1）, NucleofectorTM-treated group （group 2） and EGFP-N1 plasmids ± NucleofectorTM treated group （group 3）. Electrotransfection was performed with the U-023 program. The positive rate of ated-β-gal （SA-β-gal） staining was calculated. The expression of p16 mRNA and P16 protein were examined using RT-PCR and Western blotting, respectively. Results The positive rate of SA-β-gal staining in group 1 （9.01 ± 0.65） % was significantly lower than those in group 2 （32.00 - 0.31 ） % and group 3 （31.13 - 1.62）% （both P〈0.01）, and there was no significant difference in the latter two groups （P〉0.05）. RT-PCR and Western Blotting analysis showed that after electrotransfection, the expression 05 p 16 mRNA and P16 protein in group 2 and 3 were significantly higher than in group 1 （both P〈0.05） ; however, there was no significant difference between the latter two groups （P〉0.05）. Conclusion NucleofectorTM electrotransfection may induce immediate senescence in cultured human embryo lung fibroblasts （MRC-5 cell line）, which is probably associated with up-regulation of P16.
出处
《上海医学》
CAS
CSCD
北大核心
2009年第4期308-311,共4页
Shanghai Medical Journal
基金
国家973项目(2006CB503803)
国家自然科学基金杰出青年基金(30725036)资助项目
关键词
电转染
衰老
P16
Eletrotransfection
Senescence
P16
