摘要
目的：应用小干扰RNA（siRNA）技术抑制裸鼠喉癌皮下移植瘤人端粒酶逆转录酶（hTERT）基因表达，探讨移植瘤中增殖细胞核抗原（PCNA）及p53蛋白表达的变化。方法：根据hTERT cDNA序列构建表达短发夹RNA（shRNA）的、靶向hTERT mRNA的真核表达质粒pshRNA，其载体质粒含荧光素报告基因。建立人喉鳞状细胞癌Hep-2细胞株裸鼠皮下接种模型，将pshRNA转染入荷瘤裸鼠瘤体内，观察肿瘤生长情况。以激光共聚焦显微镜观察质粒在瘤体内的表达；以免疫组织化学SP法检测PCNA及p53蛋白在肿瘤内的表达。结果：所有裸鼠均接种成功，5d后可见皮下肿瘤形成，14d左右肿瘤直径达5～7mm。pshRNA及空质粒载体转染入瘤体后，共聚焦显微镜下见癌组织中有绿色荧光表达。病理学检查发现：pshRNA组肿瘤生长受到抑制，细胞分裂少见，可见大量癌细胞坏死。与注射生理盐水的对照组比较，转染质粒完毕后7d抑瘤率为76．50％，P〈0．01。pshRNA治疗后瘤体内PCNA蛋白表达显著下调（P〈0．05），p53蛋白表达显著上调（P〈0．05）。结论：靶向hTERT mRNA的shRNA可显著抑制人喉癌裸鼠移植瘤的生长，其机制可能是诱导PCNA下调，促进p53蛋白表达所致。
Objective：To investigate the expressions of p53 and PCNA by inhibition of hTERT with siRNA in xenografted laryngeal squamous carcinoma in nude mice. Method： pshRNA involved fluorescein gene was synthesized according to the sequence of hTERT cDNA, The xenogrfted tumor in nude mice was established by using human laryngeal squamous carcinoma cell line （Hep-2）, PshRNA packaged by METAFECTENE was transfected into the transplantation tumors, The volumes of the subcutaneous tumor were determined, Fluorescence expression in the tumors was detected by confocal microscopy. The expressions of p53 and PCNA were detected using immunohistochemistry, Result： The subcutaneous tumors appeared in all the nude mice after transplantation five days later, The diameter of subcutaneous tumors become 5～7 mm after transplantation, Many green fluorescent cells were observed by confocal microscopy after transfection of pshRNA and blank plasmid. The growth of laryngeal squamous carcinoma in nude mice was significantly decreased as compared with the control group. The proliferation activity was inhibited by 76.50%, The expressions of p53 was upregulated and the expression of PCNA was downregulated. Conclusion：The transfection of pshRNA could significantly induce inhibitory effects on growth of laryngeal squamous carcinoma in nude mice and the expression of PCNA could be effectively downregulated. The inhibition of telomerase reverse transcriptase （hTERT） mRNA could upregulate the expression of p53 and promote the apoptosis of carcinoma cells as well as inhibit the proliferation of laryngeal squamous carcinoma cells.
出处
《临床耳鼻咽喉科杂志》
CSCD
北大核心
2006年第6期264-267,共4页
Journal of Clinical Otorhinolaryngology
