纳米载体介导沉默 Survivin 基因对鼻咽癌细胞增殖凋亡及放疗敏感性的影响

摘要

目的：探讨纳米载体转染沉默鼻咽癌CNEII细胞中Survivin基因后，CNEII细胞增殖、凋亡及对放疗敏感性的改变。方法：制备聚乙烯亚胺（ PEI）-四氧化三铁（ Fe3 O4）磁性纳米粒，用PEI-Fe3 O4磁性纳米粒包被miRNA-Survivin质粒（纳米干扰质粒），将CNEII细胞分为空白对照组（不加质粒，C组）、加纳米干扰质粒组（ S组）及加miRNA-NC对照质粒组（ NC组）；3组细胞转染48 h后，荧光显微镜下观察质粒所带绿色荧光蛋白EG-FP的表达，进行细胞计数，计算转染效率；利用Real Time RT-PCR及Western Blot方法检测Survivin mRNA及蛋白表达抑制情况；转染24、48、72及96 h时用MTT法检测3组细胞的增殖情况，转染48 h时用放射线照射3组，采用流式细胞术检测3组细胞放射线照射前后的细胞凋亡率。结果：转染48 h后，加入包被miRNA-Survivin质粒的PEI-Fe3 O4磁性纳米粒的CNEII细胞转染效率为（60.9±3.9）％、mRNA表达抑制率为96.5％、蛋白表达抑制率为82.5％，均高于C组和NC组（P＜0.05），说明构建PEI-Fe3O4磁性纳米粒成功；MTT结果显示48 h以后S组细胞的吸光度值低于NC组和C组，说明细胞增殖被抑制；转染48~72 h CNEII细胞施以6Gy的放射线照射，射线照射后S组细胞凋亡率高于S组照射前（ P＜0.05），S组细胞凋亡率高于NC组和C组（ P＜0.05）。结论：鼻咽癌CNEII细胞沉默Survivin基因后的细胞的增殖被抑制、凋亡数增加，放射线照射凋亡更明显。%[ Abstract]Objective:To investigate the effects of Silencing Survivin gene via nanoparticles tech-nique on proliferation,apoptosis and radiosensitivity of nasopharyngeal carcinoma cells( CNEII ). Methods:PEI-Fe3 O4 magnetic nanoparticles were prepared. miRNA-Survivin plasmid was coated with PEI-Fe3 O4 magnetic nanoparticles. CNEII cells were divided into blank control group( without plas-mid,group C),nanoparticle interfering plasmid group( group S)and miRNA-NC plasmid control group( group NC ). Fluorescence microscope was used to measure efficiency of transfection after 48 hours. Survivin protein was detected by Western blot and mRNAs were detected by RT-PCR. Cell pro-liferation was detected by MTT 24 ,48 ,72 ,96 h after transfection;all groups were exposed to X ray after transfection at 48 h and cell apoptosis was analyzed by flow cytometry. Results:The transfection rate of miRNA-Survivin plasmid coated with PEI-Fe3O4 nanoparticles into CNEII cells was(60. 9 ± 3. 9 )%. The suppression ratio of Survivin protein and mRNA was 82 . 5% and 96 . 5% respectively in group S,which was lower than that of group C and group NC,indicating that PEI-Fe3 O4 was success-fully constructed. MTT showed that absorbance value of group S was lower than that in group C and group NC,which indicating that the proliferation of CNEII was inhibited. The ratio of cell apoptosis of group S exposed to 6 Gy was higher. The difference of Group S with Group NC and Group C was signif-icant( P<0. 05 ). Conclusion:Proliferation of transfected cells is inhibited while apoptosis rate is in-creased and even more obvious when exposed to radiation.