超声介导微泡破裂法促进外源性基因在小鼠NIH3T3细胞中的表达

Ultrasound-mediated microbubble destruction enhances exogenous gene expression in NIH3T3 cells in vitro

目的观察超声介导微泡破裂法对目的基因重组人骨形态发生蛋白-2(hBMP-2)转染靶细胞NIH3T3效率的影响。方法复苏NIH3T3细胞并传至3～4代,待细胞生长状态良好后接种至六孔培养板;将六孔板中细胞随机分为2组,分别采用质粒DNA和脂质体法(脂质体组),质粒DNA、超声和微泡法(超声介导微泡破裂组)转染目的基因;转染24～48h后在荧光倒置显微镜下对各组细胞分别计数以计算转染效率,并采用ELISA实验测定转染后hBMP-2蛋白质量浓度;使用SPSS11.5软件包分析实验结果。结果脂质体组的转染效率为(7.30±1.58)%,超声介导微泡破裂组为(11.77±3.16)%(P<0.05);脂质体组转染后hBMP-2蛋白质量浓度为(1164.35±724.67)pg/mL,超声介导微泡破裂组为(2932.70±656.27)pg/mL(P<0.05)。结论超声介导微泡破裂法可以明显提高外源性基因hBMP-2在体外NIH3T3细胞中的转染效率和蛋白质量浓度,可以为牙周再生基因治疗提供一种新型基因转移系统。

Objective To investigate the transfection efficiency of the recombinant human bone morphogenetic protein-2 （hBMP-2） gene in targeted cells by ultrasound-mediated microbubble destruction. Methods NIH3T3 cells＇ anabiosis was completed and went down to the 3rd or 4th generation, and cultured in 6 well plates. The cells were divided into 2 groups： Plasmid DNA and LipofectamineTM 2000 group （liposome group）, plasmid DNA and ultrasound and microbubble group （ultrasound-mediated microbubble destruction group）. Plasmid DNA was transfected into cells with liposome or ultrasound and microbubble. 24-48 hours later, the transfection efficiency and the concentrations of hBMP-2 were measured with fluuresence microscope and enzyme-linked immunosorbent assay （ELISA） respectively. The data were analyzed by curve fitting and t-test of SPSS 11.5. Results The transfection efficiency rate was （7.30±1.58）% in liposome group, compared with （11.77±3.16）% in ultrasound-mediated microbubble destruction group（P〈 0.05）. The concentration of hBMP-2 after transfection was （1 164.35±724.67） pg/mL in liposome group, versus （2 932.70±656.27） pg/mL in ultrasound-mediated microbubble destruction group （P〈0.05）. Conclusion Ultrasound- mediated microbubble destruction could significantly improve the transfection efficiency and expression of hBMP-2 gene in NIH3T3 cells. It may provide a new and effective gene delivery system for gene therapy in periodontal regeneration.