SPIO-PLL-pshRNA分子探针转染卵巢癌SKOV3细胞及MR成像研究

Tansfection of ovarian cancer SKOV3 cells with SPIO-PLL-pshRNA molecular probes and MR imaging

摘要:

目的：利用多聚赖氨酸修饰的超顺磁性氧化铁（superparamagnetic iron oxide modified with Poly-L-lysine，SPIO-PLL）及质粒pGenesil1-shRNA构建SPIO-PLL-pshRNA分子探针，并初步评价其体外转染卵巢癌细胞SKOV3及MR成像的可行性。方法：利用静电吸附法连接SPIO-PLL和质粒pGenesil1-shRNA，微量分光光度计检测复合物离心后上清液中质粒DNA浓度，评测SPIO-PLL与质粒的结合能力；凝胶阻滞实验及zeta电位测定验证两者结合；通过普鲁士蓝染色法观察铁的入胞情况及荧光显微镜观察绿色荧光蛋白在细胞中的表达来检测复合物的细胞转染能力；MR扫描观察细胞转染后信号改变。结果：DNA结合实验、凝胶阻滞实验结果显示当SPIO-PLL和质粒质量比达6∶1时，二者可有效结合；激光粒度仪测定SPIO、SPIO-PLL、SPIO-PLL-pshRNA 3组样品的zeta电位分别为（-14.8±0.35）、（15.2±0.55）、（3.6±0.35） mV，3组结果差异有统计学意义（F=6 323.782，P=0.000；LSD-t：均P=0.000）；所制备的SPIO-PLL-pshRNA分子探针体外转染细胞后，用普鲁士蓝染色法可检测到细胞内有铁颗粒分布，荧光显微镜观察到绿色荧光蛋白表达，MR扫描结果示无探针转染组及10、20、40 μg/ml 3种浓度探针转染组的T2*信号强度值分别为（558±19）、（427±16）、（283±19）、（191±8），琼脂糖组及蒸馏水组分别为（600±36）、（670±16），随着探针浓度升高，信号强度降低，各组结果差异有统计学意义（F=279.667，P=0.000；SNK-q：均P<0.05）。结论：SPIO-PLL纳米粒可有效结合质粒pGenesil1-shRNA，制得SPIO-PLL-pshRNA分子探针，该探针于体外成功转染卵巢癌SKOV3细胞，并于MR扫描下T2*WI信号强度降低。

Abstract:

Objective：To prepare SPIO-PLL-pshRNA molecular probes and to preliminarily evaluate the feasibility of transfecting SKOV3 cells and MR imaging in vitro. Methods：Superparamagnetic iron oxide modified with Poly-L-lysine（SPIO-PLL） was bound with plasmid pGenesil1-shRNA through electrostatic absorption. Concentration of plasmid DNA in the supernatant was detected by micro-ultraviolet spectrophotometer after the centrifugation of the complexes. By this way，the ability of plasmid DNA binding to SPIO-PLL could be evaluated. Conjunction between SPIO-PLL and plasmid DNA was confirmed via gel retardation experiments and zeta potential methods. Cell transfection was evaluated with Prussia blue staining for iron assessment and fluorescence microscope for green fluorescent protein（GFP） expression detection. Changes of cells in MR signal intensities were observed after the transfection. Results：DNA binding tests，gel retardation experiments and zeta potential methods demonstrated that SPIO-PLL could effectively bind plasmid DNA when the w/w ratio of SPIO-PLL and plasmid DNA was more than 6∶1. zeta electric potentials of SPIO，SPIO-PLL and SPIO-PLL-pshRNA were （-14.8±0.35），（15.2±0.55） mV and （3.6±0.35） mV，with statistical differences among groups（F=6323.782，P=0.000；LSD：all P=0.000）. After transfection with the SPIO-PLL-pshRNA complexes，iron particles could be detected in cells with Prussia blue staining；green fluorescence could be observed by fluorescent microscope；MR imaging showed T2*WI signal intensities reduction（558±19，427±16，283±19 and 191±8 in non transfection group，10，20 μg/ml and 40 μg/ml groups respectively；600±36 and 670±16 in agarose group and distilled water group）. The signal intensity was decreased with the increase of complex concentration and statistical differences were observed among groups（F=279.667，P=0.000；SNK-q：all P<0.05）. Conclusions：SPIO-PLL nanoparticles can effectively bind with plasmid pGenesil1-shRNA as SPIO-PLL-pshRNA molecular probe. This probe can successfully transfect SKOV3 cells and T2*WI signal intensity reduction can be observed by MR scanning.