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荧光素标记的反义寡核苷酸在大鼠肾小管上皮细胞中的时相分布

Distribution of fluorescent-labeled antisence oligodeoxynucleotide in rat kidney tubular epithelial cell

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【作者】 刘晓波余学清周树录董秀清

【Author】 LIU Xiao-bo,YU Xue-qing,ZHOU Shu-lu,et al. Department of Microbiology,Guangdong Pharmaceutical University,Guangzhou 510006,China

【机构】 广东药学院基础学院微生物学教研室中山大学附属第一医院教育部肾脏病重点实验室广州市花都区人民医院肾内科中山大学附属第一医院教育部肾脏病重点实验室 广东省广州510006

【摘要】 目的观察反义寡核苷酸(AS-ODN)在大鼠肾小管上皮细胞株(NRK52E)中的时相分布;比较AS-ODN以脂质体(DOTAP)包裹或以游离形式转染细胞的区别。方法合成一条与大鼠骨调素(OPN)cDNA序列互补的AS-ODN,在5′端标记荧光素(FAM);将AS-ODN/DOTAP复合物(AS-ODN浓度3μmol/L)或游离AS-ODN(6μmol/L)处理培养的NRK52E细胞,于不同时间点取细胞于荧光显微镜下观察计数,同时进行普通光镜下细胞计数,计算转染效率。结果AS-ODN/DOTAP复合物在细胞中呈颗粒状分布,处理细胞3h后可见细胞胞浆中出现荧光颗粒,之后AS-ODN转移进入胞核,第8h时在胞核中可达很高浓度,并维持较长时间;游离AS-ODN在细胞中呈弥散状分布,处理细胞5h后可见细胞中出现荧光,第8h时在胞浆和胞核中可达较高浓度,并维持较长时间;AS-ODN/DOTAP复合物的转染效率明显高于游离AS-ODN;细胞是否贴壁生长及贴壁生长的细胞密度也影响AS-ODN的转染效率。结论AS-ODN转染进入细胞后最终浓聚在细胞核中,并维持较长时间;阳离子脂质体能提高AS-ODN转染速率和转染效率。

【Abstract】 Objective To directly observe the distribution of fluorescent-labeled antisence oligodeoxynucleotide(AS-ODN)in cultured rat kidney epithelial cell line(NRK52E) by fluorescence microscopy and to evaluate the transfection efficiency of free AS-ODN or AS-ODN/cationic liposome complex.Methods One AS-ODN complementary to rat OPN cDNA sequences was synthesized with respect to all its nucleotides modified with phosphrothioate and the 5′ end nucleotide labeled with fluorescent(FAM);the NRK52E cells were incubated with AS-ODN/DOTAP complexes or free AS-ODN at 37 ℃,5% CO2 conditions and observed for the intracellular distribution of AS-ODN under fluorescent microscope at different time point.The transfection efficiency of AS-ODN was calculated by the ratio of positive cell number counted under fluorescent microscopy to the total cells counted under light microscopy.Results When incubated with the NRK52E cells for 3h,AS-ODN/DOTAP complex was visualized in cytoplasm of the cells in form of particles with yellow-green fluorenscence.Afterwards, the particles transferred into the cell nucleus and accumulated there with higher concentration for more than 10 h.Free AS-ODN was visualized within the cytoplasm and nucleus when incubated with the NRK52E cells for 6 h and the flurescence was in diffusion form;Free AS-ODN was still accumulated in the cell nucleus with higher concentration for several hours.AS-ODN/DOTAP complex could exhibited more higher transfection efficiency than free AS-ODN and the transfection efficiency was also influenced by many other factors such as cell density etc.Conclusion After transfection into cells,AS-ODN finally entered into cell nucleus and accumulated there for several hours.Cationic liposome could increase the speed and efficiency of AS-ODN transfection into cells.

【基金】 国家自然科学基金资助项目(39970704);广东省自然科学基金资助项目(980058,010763)
  • 【文献出处】 检验医学与临床 ,Laboratory Medicine and Clinic , 编辑部邮箱 ,2006年09期
  • 【分类号】R692
  • 【下载频次】90
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