摘要
目的探讨针对血管内皮生长因子（VEGF）、端粒酶逆转录酶（TERT）、Bcl-xl的3个短发夹状双链RNA（shRNA）的串联表达质粒对人喉鳞癌裸鼠移植瘤生长抑制作用及其机制。方法构建串联表达3个shRNA的质粒pEGFP-shVEGF—shTERT-shBcl-xl。建立人喉鳞癌Hep-2细胞系荷瘤裸鼠模型。将该质粒转染入荷瘤裸鼠瘤体内，观察肿瘤生长情况。荧光定量即时PCR检测三种目的基因的mRNA在肿瘤组织中的表达情况，Western blot法检测三种蛋白的表达，原位末端标记法（TUNEL法）检测肿瘤细胞凋亡，免疫组织化学SP法检测肿瘤组织微血管密度。结果与治疗前比较，质粒pEGFP-shVEGF—shTERT—shBcl-xl注射裸鼠皮下移植瘤后，肿瘤生长明显受抑制，治疗后14d抑瘤率达91．2％。三个目的基因的mRNA及蛋白表达水平比治疗前均有显著下降。移植瘤组织内检测到大量凋亡细胞，凋亡指数为（60．34±5．32）％。治疗组微血管密度明显低于生理盐水对照组及阴性质粒处理组。结论pEGFP—shVEGF—shTERT—shBcl-xl能高效特异性地同时抑制3个靶基因在人喉鳞癌Hep-2细胞中的表达，增强基因沉默的多样性，并显著的抑制移植瘤的生长，提示未来应用多基因共沉默治疗喉鳞癌可能具有广阔的前景。
Objective To investigate the biological impact of pEGFP-shVEGF-shTERT-shBcl-xl expression in human laryngeal squamous carcinomas xenografted in nude mice and the related antitumor mechanism. Methods A recombinant plasmid vector containing 3 different short hairpin RNA （shRNA） segments including pEGFP-shVEGF-shTERT-shBcl-xl was constructed and directly injected into the grafted tumors of human laryngeal squamous carcinoma in nude mice. The mRNA and protein expressions were determined by real-time RT-PCR and Western blot respectively. Apoptosis was determined by TUNEL assay using a commercial kit. Intratumoral microvessel density （MVD） was assessed by immunhistochemistry. Results On the 14th days after the final treatment, mRNA and protein expression of VEGF, TERT, and Bcl-xl were markedly suppressed. The tumor sizes were significantly smaller than those in the other two group, with an overall tumor inhibition ratio of 91.2%. MVD counts in the pEGFP-shVEGF-shTERT-shBcl- xl treated group were significantly lower than those of the other two groups, along with increased apoptotic cells. Conclusions The data showed that inhibition of VEGF,TERT, Bcl-xl expression by RNAi technique induces cellular apoptosis and suppresses the growth of laryngeal squamous carcinoma in vivo. VEGF,TERT and Bcl-xl may be involved in the development of laryngeal cancers. The findings suggest a synergistic tumor therapeutic effect through simultaneous inhibition of the three genes. Multi-target RNA interference may provide a powerful strategy against human laryngeal cancers.
出处
《中华病理学杂志》
CAS
CSCD
北大核心
2008年第12期831-836,共6页
Chinese Journal of Pathology
基金
国家自然科学基金资助项目（30471873,30672313,30740012）
湖北省科技攻关项目资助项目（2007AA302808）
武汉市科技局国际合作项目资助项目（200770834319）
关键词
喉肿瘤
癌
鳞状细胞
RNA干扰
动物实验
Laryngeal neoplasms
Carcinoma, squamous cell
RNA interference
Animal experimentation
