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

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。

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% CO_2 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.