卵巢癌特异性分子探针的制备及初步体外实验研究

发布时间：2018-04-10 10:17

选题：分子影像学　视角：卵巢癌　引自：《重庆医科大学》2014年硕士论文

【摘要】：目的：构建针对卵巢癌的SPIO-PLL-pshRNA分子探针，并初步评价其体外转染卵巢癌SKOV3细胞及MR成像的可行性。方法：利用静电吸附法连接SPIO-PLL复合物及质粒pGenesil1-shRNA，微量分光光度计检测不同比例SPIO-PLL-pshRNA复合物离心后上清液中游离质粒浓度，评测SPIO-PLL与质粒的结合能力，确定制备SPIO-PLL-pshRNA分子探针的最佳质量比；凝胶阻滞实验及zeta电位测定进一步验证探针中SPIO-PLL与质粒的结合情况；CCK-8法检测探针的细胞急性毒性；普鲁士蓝染色法初步观察探针转染细胞情况；荧光显微镜观察绿色荧光蛋白在细胞中的表达来进一步评价探针的细胞转染能力；透射电子显微镜观察探针中SPIO在细胞中的部位；MR扫描观察探针转染细胞后信号强度的改变。结果：DNA结合实验、凝胶阻滞实验结果显示，当SPIO-PLL复合物和质粒pGenesil1-shRNA的质量比达6：1时，二者可有效结合；激光粒度仪测定SPIO、SPIO-PLL、SPIO-PLL-pshRNA三组样品的zeta电位分别为（-14.8±0.35）mV、（15.2±0.55）mV、（3.6±0.35）mV，三组结果差异有统计学意义（ANOVA：F=6323.782，P=0.000；LSD：均P=0.000）；细胞毒性试验结果显示，，在探针浓度低于50mg/L的范围内，本研究由国家自然科学基金资助（编号：81171366）细胞存活率高于80%；所制备的SPIO-PLL-pshRNA分子探针体外转染卵巢癌SKOV3细胞后，普鲁士蓝染色法可检测到细胞内有铁颗粒分布，荧光显微镜观察细胞内有绿色荧光蛋白表达，透射电子显微镜下见到探针中的铁颗粒存在于细胞胞浆内；MR成像结果示，探针转染细胞后T2*信号强度明显降低，且随着探针浓度升高，信号强度越低，各浓度组T2*信号强度差异有统计学意义（ANOVA：F=279.667，P=0.000；SNK：均P0.05）。结论：成功制备针对卵巢癌的特异性分子探针，该探针在体外能成功转染卵巢癌SKOV3细胞，对细胞毒性小，且能被MR扫描的T2＊WI序列敏感检测。
[Abstract]:Aim: to construct a SPIO-PLL-pshRNA molecular probe for ovarian cancer and to evaluate the feasibility of transfection of ovarian cancer SKOV3 cells and Mr imaging in vitro.Methods: the SPIO-PLL complex and plasmid pGenesil1-shRNAs were connected by electrostatic adsorption. The concentration of free plasmid in supernatant of different proportion of SPIO-PLL-pshRNA complex after centrifugation was detected by microspectrophotometer, and the binding ability of SPIO-PLL to plasmid was evaluated.The optimal mass ratio of SPIO-PLL-pshRNA molecular probes was determined, and the binding of SPIO-PLL to plasmids in the probes was further verified by gel block assay and zeta potential determination. CCK-8 method was used to detect the acute cytotoxicity of the probes.Prussian blue staining was used to observe the transfection of the probe, and the expression of green fluorescent protein in the cells was observed by fluorescence microscope to further evaluate the transfection ability of the probe.The position of SPIO in the probe was observed by transmission electron microscope (TEM). Mr scanning was used to observe the change of signal intensity after transfection of the probe.Results the results of DNA binding test and gel block test showed that SPIO-PLL complex and plasmid pGenesil1-shRNA could be effectively combined at 6:1 when the mass of Prida was 6:1.In this study, the survival rate of the cells funded by the National Natural Science Foundation of China (No. 81171366) was higher than that of 80%. After transfection of the SPIO-PLL-pshRNA molecular probe into ovarian cancer SKOV3 cells in vitro, Prussian blue staining could detect the distribution of iron particles in the cells.The expression of green fluorescent protein was observed by fluorescence microscope. The results of Mr imaging showed that the iron particles in the probe were present in the cytoplasm of the cells. The signal intensity of T2 * was significantly decreased after the probe was transfected into the cells.With the increase of probe concentration, the signal intensity decreased, and the difference of signal intensity of T2 * in each concentration group was statistically significant (P < 0.05).Conclusion: a specific molecular probe for ovarian cancer was successfully prepared. The probe can be successfully transfected into ovarian cancer SKOV3 cells in vitro, which has little cytotoxicity and can be detected by Mr scanning T2*WI sequence sensitively.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.31

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