【摘要】 目的研究超声微泡介导ABCB1a基因siRNA质粒转染L2RYC细胞的效率以及对ABCB1a水平和P-糖蛋白(P glycoprotein,P-gp)表达的影响。方法制备脂质微泡;设计4对siABCB1a序列,并构建4种真核表达质粒pSES-siABCB1a;转染L2RYC细胞(单纯质粒组、质粒+微泡组、质粒+超声组、质粒+微泡+超声组),另设空白对照组。采用流式细胞术检测质粒的转染效率;实时荧光定量PCR及Western blot检测转染后L2RYC细胞中ABCB1a基因及P-gp的表达。结果 4种pSES-siABCB1a质粒经PCR、双酶切及测序证实构建正确;质粒+微泡+超声组pSES-siABCB1a质粒可高效转染L2RYC细胞,可见荧光表达,转染效率为(49.35±5.89)%;转染后ABCB1a基因mRNA及P-gp的表达均明显降低(P<0.05)。结论超声微泡可促进外源基因体外转染L2RYC细胞,siABCB1a能有效抑制ABCB1a基因和P-gp的表达。更多还原

【Abstract】 Objective To investigate the transfection efficacy of ultrasound-microbubble mediated siRNA plasmid of ATP-binding cassette sub-family B member 1a(ABCB1a) gene as well as its effect on the expressions of ABCB1a and P glycoprotein(P-gp).Methods Lipid microbubbles were prepared.Four pairs of siABCB1a sequences were designed,based on which four kinds of plasmid pSES-siABCB1a were constructed and transfected to L2RYC cells in various groups(plasmid,plasmid + microbubble,plasmid + ultrasound,plasmid + microbubble + ultrasound).A blank control group was set up,in which the cells were untransfected.The transfection efficacy was determined by flow cytometry.The expressions of ABCB1a gene and P-gp in transfected L2RYC cells were determined by real-time fluorescent quantitative PCR and Western blot.Results PCR,restriction analysis and sequencing proved that the four kinds of plasmid pSES-siABCB1a were constructed correctly.Fluorescence was observed in L2RYC cells in plasmid + microbubble + ultrasound group,with a transfection efficacy of(49.35 ± 5.89)%.Both the expression levels of ABCB1a mRNA and P-gp decreased significantly after transfection(P < 0.05).Conclusion Ultrasound-microbubbles promoted the in vitro transfection of L2RYC cells with exogenous gene,while siABCB1a effectively inhibited the expressions of ABCB1a gene and P-gp.更多还原