RNA同时干扰四个不同基因对鼻咽癌细胞CNE-2Z生长增殖的实验研究  

Effects of RNA interference targeting four different genes on the growth and proliferation of nasopharyngeal carcinoma CNE-2Z cells

作　　者：宋英[1] 董明敏[1] 杨海峰 

机构地区：[1]郑州大学第一附属医院耳鼻咽喉科,450052 [2]郑州市管城区疾病预防控制中心

出　　处：《中华耳鼻咽喉头颈外科杂志》2010年第9期751-758,共8页Chinese Journal of Otorhinolaryngology Head and Neck Surgery

摘　　要：目的 应用RNA干扰技术构建一个载体同时编码四个基因,即血管内皮生长因子（VEGF）、致癌基因蛋白（c-myc）、存活素（survivin）和端粒酶反转录酶的短发夹重组质粒,并与单基因质粒做对比,通过体内外实验,研究其对鼻咽癌细胞CNE-2Z生长增殖的影响.方法 构建同时表达VEGF、c-myc、survivin和端粒酶反转录酶并含荧光素标记的短发夹RNA质粒命名为P-1,并构建单独表达VEGF、c-myc、survivin和端粒酶反转录酶的质粒,分别命名为P-2、P-3、P-4和P-5,分别将其将转染人鼻咽癌CNE-2Z细胞株及荷瘤裸鼠瘤体内.以激光共聚焦显微镜观察质粒在CNE-2Z细胞及瘤体内的转染及表达情况.四甲基偶氮噻唑蓝法检测细胞增殖活性,实时PCR在mRNA水平上检测对目的基因表达的抑制作用,免疫印迹法在蛋白水平上检测对目的基因的封闭效果,Trans well侵袭小室模型观察导入CNE-2Z细胞对其恶性生物学行为的改变,流式细胞仪观察各组凋亡率,体内裸鼠成瘤实验观察质粒对肿瘤的抑制效果.结果 多基因干扰质粒转染鼻咽癌细胞后,CNE-2Z细胞增殖受到明显抑制,体外侵袭能力显著下降（P值均＜0.05）.实时PCR证实多基因组4个不同基因mRNA表达均降低,免疫印迹技术验证目的基因蛋白同时表达下调.流式细胞仪结果证明多基因凋亡率明显高于单基因组（P值均＜0.05）.体内抑瘤实验表明,与阴性组和空白组相比,P-1组移植瘤生长明显受到抑制（P＜0.05）,并且多基因质粒抑制肿瘤细胞增长的作用要强于单基因干扰质粒（户值均＜0.05）.结论 同时干扰多个基因能有效抑制鼻咽癌细胞增殖并诱导其凋亡,为鼻咽癌基因治疗奠定了良好的实验基础.Objective To explore the effects of RNA interference （RNAi） targeting four different genes （VEGF, c-myc, survivin, hTERT） on the growth and proliferation of nasopharyngeal carcinoma （NPC） CNE-2Z cells. Methods Plasmid-1 targeted all four genes, plasmid-2, 3, 4 and 5 targeted VEGF,c-myc, survivin and hTERT respectively. These plasmids were transfected seperately into human NPC CNE-2Z cells and xenograft tumors in nude mice. The expressions of plasmids in NPC CNE-2Z cells and xenograft tumors were observed. Cell proliferation was detected with MTT assay. The inhibitory effects on target genes were evaluated with RT-PCR and Western blot respectively. The effects of the plasmids on the biological behavior of CNE-2Z cells were observed with Transwell invision chamber model. Apoptosis was determined with flow cytometer. The inhibitory effect of the plasmids on xenograft tumors were observed in nude mice.Results CNE-2Z cell proliferation was significantly inhibited and in vitro invasion ability was significantly decreased in the plasmid-1 group compared with those in the plasmid 2 -5 groups （all P 〈 0. 05 ）. mRNAand protein expressions of all four genes decreased in the plamid-1 group. The apoptosis rate in the plamid1 group was higher than that in the plasmid 2 - 5 groups （ all P 〈 0. 05 ）. Growth of xenograft tumors in nude mice were significantly inhibited in the plasmid 1 -5 groups, particularly in the plasmid-1 group.Conclusion RNA interference targeting multiple genes can effectively inhibit NPC proliferation and induce apoptosis, which provides an experiment basis for NPC gene therapy.

关 键 词：RNA干扰 基因疗法 鼻咽肿瘤 细胞系 肿瘤 细胞增殖 

分 类 号：R739.63[医药卫生—肿瘤]