選題：分子影像學　視角：卵巢癌　引自：《重慶醫(yī)科大學》2014年碩士論文

【摘要】：目的：構(gòu)建針對卵巢癌的SPIO-PLL-pshRNA分子探針，并初步評價其體外轉(zhuǎn)染卵巢癌SKOV3細胞及MR成像的可行性。 方法：利用靜電吸附法連接SPIO-PLL復合物及質(zhì)粒pGenesil1-shRNA，微量分光光度計檢測不同比例SPIO-PLL-pshRNA復合物離心后上清液中游離質(zhì)粒濃度，評測SPIO-PLL與質(zhì)粒的結(jié)合能力，確定制備SPIO-PLL-pshRNA分子探針的最佳質(zhì)量比；凝膠阻滯實驗及zeta電位測定進一步驗證探針中SPIO-PLL與質(zhì)粒的結(jié)合情況；CCK-8法檢測探針的細胞急性毒性；普魯士藍染色法初步觀察探針轉(zhuǎn)染細胞情況；熒光顯微鏡觀察綠色熒光蛋白在細胞中的表達來進一步評價探針的細胞轉(zhuǎn)染能力；透射電子顯微鏡觀察探針中SPIO在細胞中的部位；MR掃描觀察探針轉(zhuǎn)染細胞后信號強度的改變。 結(jié)果：DNA結(jié)合實驗、凝膠阻滯實驗結(jié)果顯示，當SPIO-PLL復合物和質(zhì)粒pGenesil1-shRNA的質(zhì)量比達6：1時，二者可有效結(jié)合；激光粒度儀測定SPIO、SPIO-PLL、SPIO-PLL-pshRNA三組樣品的zeta電位分別為（-14.8±0.35）mV、（15.2±0.55）mV、（3.6±0.35）mV，三組結(jié)果差異有統(tǒng)計學意義（ANOVA：F=6323.782，P=0.000；LSD：均P=0.000）；細胞毒性試驗結(jié)果顯示，，在探針濃度低于50mg/L的范圍內(nèi)，本研究由國家自然科學基金資助（編號：81171366）細胞存活率高于80%；所制備的SPIO-PLL-pshRNA分子探針體外轉(zhuǎn)染卵巢癌SKOV3細胞后，普魯士藍染色法可檢測到細胞內(nèi)有鐵顆粒分布，熒光顯微鏡觀察細胞內(nèi)有綠色熒光蛋白表達，透射電子顯微鏡下見到探針中的鐵顆粒存在于細胞胞漿內(nèi)；MR成像結(jié)果示，探針轉(zhuǎn)染細胞后T2*信號強度明顯降低，且隨著探針濃度升高，信號強度越低，各濃度組T2*信號強度差異有統(tǒng)計學意義（ANOVA：F=279.667，P=0.000；SNK：均P0.05）。 結(jié)論：成功制備針對卵巢癌的特異性分子探針，該探針在體外能成功轉(zhuǎn)染卵巢癌SKOV3細胞，對細胞毒性小，且能被MR掃描的T2＊WI序列敏感檢測。
[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.
【學位授予單位】：重慶醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R737.31

【參考文獻】

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2 喬瑞瑞;賈巧娟;曾劍峰;高明遠;;磁性氧化鐵納米顆粒及其磁共振成像應用[J];生物物理學報;2011年04期

3 楊華;張小明;邵陽;蔣紅;曾南林;;超順性氧化鐵在細胞內(nèi)外對磁共振信號的影響[J];中國組織工程研究與臨床康復;2008年30期

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