氮磷比对PEI介导BMP-7基因转染骨髓间充质干细胞的影响

目的:研究氮磷比(N/P值)对非病毒基因载体聚乙烯亚胺(polyethylenimine,PEI)介导骨形态发生蛋白-7(bone morphogenetic protein-7,BMP-7)基因转染骨髓间充质干细胞(bone mesenchymal stem cells,MSC)的影响,优化PEI/DNA复合物制备参数,为其应用于骨组织工程提供实验基础.方法:制备不同N/P值的含BMP-7基因的PEI/DNA复合物,测定复合物的粒径、Zeta电位以及PEI保护DNA抵御DNA酶消化的能力,检测复合物对MSC细胞的毒性以及转染表达BMP-7蛋白量.结果:当N/P值在3-30范围时,形成微粒粒径稳定在100 ～ 150 nm;当N/P值在5～ 30范围时,Zeta电位稳定在30 ～ 40 mV.当N/P值＞3时,随着PEI的增加,PEI对DNA的保护功能增强.当N/P值为7～10时,可以获得最高的基因转染效率.当N/P值大于10时,细胞毒性明显增加.结论:N/P值为7～ 10时,含BMP-7基因的PEI/DNA复合物细胞毒性小,转染效率高.

Effects of N/P ratios on PEI-mediated BMP-7 gene expression in bone mesenchymal stem cells

Objective: To study the influence of N/P ratios on polyethylenimine(PEI)-mediated bone morphogenetic protein-7(BMP-7) gene expression in bone mesenchymal stem cells(MSC),and optimize the fabrication parameters of PEI/DNA complexes.Methods: PEI/DNA complexes containing BMP-7 gene were prepared with various values of N/P ratio,the size and zeta potential of PEI/DNA complexes were measured.PEI’s protective effects on DNA against enzymatic digestion were determined,and the cytotoxicity of PEI/DNA nanoparticles and the expression of BMP-7 in transfected MSCs were evaluated.Results: When the N/P ratios were at the range of 3-30,the diameter of nanoparticles was 100-150 nm.At the N/P value of 5-30,the Zeta potential of complexes was 30-40 mV.PEI showed desired protective effects on DNA against enzymatic digestion by Dnase when N/P values of the complexes were above 3.At the N/P value of 7-10,the gene expression of BMP-7 reached its highest level.Conclusion: When the N/P ratios are at the range of 7-10,PEI/DNA complexes show lower cytotoxicity and higher transfection efficiency.