邓雯,孙茂钢,赵艳,赵厚育
DENG Wen,SUN Maogang,ZHAO Yan,ZHAO houyu

• 1:贵州医科大学
• 2:贵州医科大学分子生物学重点实验室
• 3:贵州医科大学附院耳鼻咽喉科

摘要(Abstract)：

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(Fe3O4)磁性纳米粒,用PEI-Fe3O4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EGFP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-Fe3O4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。
Objective: To investigate the effects of Silencing Survivin gene via nanoparticles technique on proliferation,apoptosis and radiosensitivity of nasopharyngeal carcinoma cells( CNEII).Methods: PEI-Fe3O4 magnetic nanoparticles were prepared. miRNA-Survivin plasmid was coated with PEI-Fe3O4 magnetic nanoparticles. CNEII cells were divided into blank control group( without plasmid,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 proliferation 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-Fe3O4 was successfully 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 significant( P < 0. 05). Conclusion: Proliferation of transfected cells is inhibited while apoptosis rate is increased and even more obvious when exposed to radiation.

关键词(KeyWords)： 鼻咽癌;Survivin基因;细胞增殖;细胞凋亡;放疗敏感性
nasopharyngeal carcinoma;Survivin gene;cell proliferation;apoptosis;radiosensitivity

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金地区科学基金项目(NO.81260353)

作者(Author): 邓雯,孙茂钢,赵艳,赵厚育
DENG Wen,SUN Maogang,ZHAO Yan,ZHAO houyu

DOI: 10.19367/j.cnki.1000-2707.2016.04.003

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